INTRODUCTION: The intervertebral disc is the largest avascular, low nutrient organ. Autophagy is an important cell survival mechanism by self-digestion and recycling damaged components under stress, primarily nutrient deprivation. We hence hypothesized that resident cells would utilize autophagy to cope with the harsh disc environment. However, pharmacological autophagy inhibitors elicit effects through modulating intracellular signaling or lysosomal acidification, thereby including multiple confounders. Our objective was to clarify specific roles of autophagy in in-vitro human and in-vivo rat disc homeostasis through RNA interference (RNAi)-mediated knockdown of autophagy-related gene 5 (Atg5), essential for autophagy.

METHODS: In human disc nucleus pulposus cells collected during lumbar interbody fusion surgery (n = 25: age, 63.0 ± 5.3 years old; 8 males and 17 females; Pfirrmann degeneration grade, 3.4 ± 0.2), autophagy inhibition by RNAi of Atg5 was tested. Cell viability with levels of autophagy including Atg5 expression, apoptosis, and senescence was assessed under serum starvation and/or pro-inflammatory interleukin-1 beta (IL-1β) stimulation. In rat tail disc tissues (n = 28, 12 weeks old, all males), autophagy was monitored following Alexa Fluor® 555-labeled Atg5-small interfering RNA (siRNA) injection. Furthermore, radiographical and histological disruption with the incidence of apoptosis and senescence induced by 24-h temporary static compression at 1.3 MPa was observed for 56-d time-course. The paired t-test or one-way repeated measures or two-way analysis of variance with the Tukey–Kramer post-hoc test was used.

RESULTS: [In-vitro study] In human disc cells, Western blotting showed that serum deprivation and IL-1β stimulation increased autophagy marker LC3-II and decreased autophagy substrate p62/SQSTM1, indicating enhanced autophagy. Then, multiple Atg5 siRNAs with different sequences consistently suppressed autophagy through decreases in Atg5 protein (26.8–27.4%, P < 0.0001). Cell viability was kept by Atg5 RNAi in serum-supplemented media (95.5%, P = 0.28) but reduced in serum-free media (80.4%, P = 0.0013) with IL-1β (69.9%, P = 0.0008). Moreover, Atg5 RNAi accelerated IL-1β-induced increases in expression of apoptosis and senescence markers in Western blotting, TUNEL staining, and SA-β-gal staining. Meanwhile, Atg5 RNAi less affected IL-1β-induced catabolic MMP-3 and MMP-13 release, down-regulated anabolic aggrecan and Col2a1 gene expression, and MAPK-pathway activation. [In-vivo study] In rat tail discs, immunofluorescence detected intradiscal signals for the Alexa Fluor® 555-labeled Atg5 siRNA at 2-d, 28-d, and even 56-d post-injection. Western blotting also found 2-d, 28-d, and 56-d autophagy suppression with Atg5 knockdown (P = 0.003–0.03). Under 24-h temporary static compression, Atg5 siRNA-injected loaded discs presented up to 56 d radiographic height loss (P = 0.001–0.03), histomorphological damage (P = 0.0003–0.002), and increased immunofluorescent positivity for apoptotic TUNEL and senescent p16/INK4a (P < 0.0001).

DISCUSSION: This in-vitro and in-vivo loss-of-function study findings suggests that Atg5-dependent autophagy primarily protects against human and rat disc cellular apoptosis and senescence rather than extracellular matrix catabolism. Then secondarily, enhanced cell survival by autophagy could favorably contribute to the maintenance of disc homeostasis under long-term stress conditions. Autophagy is thus a potent new molecular therapeutic target for intervertebral disc disease.