Involvement of autophagy in the maintenance of rat intervertebral disc homeostasis: an in-vitro and in-vivo RNA interference study of Atg5

ObjectiveIn the largest avascular low-nutrient intervertebral disc, resident cells would utilize autophagy, a stress-response survival mechanism by self-digestion and recycling wastes. Our goal was to elucidate the involvement of autophagy in disc homeostasis through RNA interference of autophagy-related gene 5 (Atg5).DesignIn vitro, small interfering RNAs (siRNAs) targeting autophagy-essential Atg5 were transfected into rat disc cells. 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 vivo, time-course autophagic flux was monitored following Alexa Fluor® 555-labeled Atg5-siRNA injection into rat tail discs. Furthermore, 24-h temporary static compression-induced disruption of Atg5 siRNA-injected discs was observed by radiography, histomorphology, and immunofluorescence.ResultsIn disc cells, three different Atg5 siRNAs consistently suppressed autophagy with Atg5 protein knockdown (mean 44.4% [95% confidence interval: −51.7, −37.1], 51.5% [−80.5, −22.5], 62.3% [−96.6, −28.2]). Then, Atg5 knockdown reduced cell viability through apoptosis and senescence not in serum-supplemented medium (93.6% [−0.8, 21.4]) but in serum-deprived medium (66.4% [−29.8, −8.6]) further with IL-1β (44.5% [−36.9, −23.5]). In disc tissues, immunofluorescence detected intradiscal signals for the labeled siRNA even at 56-d post-injection. Immunoblotting found 56-d autophagy suppression with prolonged Atg5 knockdown (33.2% [−52.8, −5.3]). With compression, Atg5 siRNA-injected discs presented radiographic height loss ([−43.9, −0.8]), histological damage ([−5.5, −0.2]), and immunofluorescent apoptosis ([2.2, 22.2]) and senescence ([4.1, 19.9]) induction compared to control siRNA-injected discs at 56 d.ConclusionsThis loss-of-function study suggests Atg5-dependent autophagy-mediated anti-apoptosis and anti-senescence. Autophagy could be a molecular therapeutic target for degenerative disc disease.