Inhibition of Autophagy at Different Stages by ATG5 Knockdown and Chloroquine Supplementation Enhances Consistent Human Disc Cellular Apoptosis and Senescence Induction rather than Extracellular Matrix Catabolism

Abstract

The intervertebral disc is the largest avascular organ. Autophagy is an important cell survival mechanism by self-digestion and recycling damaged components under stress, primarily nutrient deprivation. Resident cells would utilize autophagy to cope with the harsh disc environment. Our objective was to elucidate the roles of human disc cellular autophagy. In human disc cells, serum deprivation and pro-inflammatory interleukin-1β (IL-1β) stimulation increased autophagy marker microtubule-associated protein 1 light chain 3 (LC3)-II and decreased autophagy substrate p62/sequestosome 1 (p62/SQSTM1), indicating enhanced autophagy. Then, RNA interference (RNAi) of autophagy-related gene 5 (ATG5), essential for autophagy, showed decreases in ATG5 protein (26.8%–27.4%, p < 0.0001), which suppressed early-stage autophagy with decreased LC3-II and increased p62/SQSTM1. Cell viability was maintained 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 changes in apoptosis and senescence. Meanwhile, ATG5 RNAi unaffected IL-1β-induced catabolic matrix metalloproteinase release, down-regulated anabolic gene expression, and mitogen-activated protein kinase pathway activation. Lysosomotropic chloroquine supplementation presented late-stage autophagy inhibition with apoptosis and senescence induction, while catabolic enzyme production was modest. Disc-tissue analysis detected age-related changes in ATG5, LC3-II, and p62/SQSTM1. In summary, autophagy protects against human disc cellular apoptosis and senescence rather than extracellular matrix catabolism.

Involvement of Autophagy in Rat Tail Static Compression-Induced Intervertebral Disc Degeneration and Notochordal Cell Disappearance

“…In vitro, serum deprivation-induced autophagy in disc NP and AF cells is consistent with prior reports [ 19 , 20 , 23 , 41 ]. To mimic endplate closure and subsequent cut off of serum supply, in vitro responses to limited nutrition were observed.…”

“…A Western blotting study of human degenerative disc tissues surgically collected found peaked LC3-II protein expression in Pfirrmann [ 43 ] grade 3 compared to grades 2, 4, and 5 as well as severity-dependent increases in p62/SQSTM1 expression [ 45 ]. We also studied autophagy-related ATG5, LC3-II, and p62/SQSTM1 protein expression in human lumbar disc surgical specimens and observed age-dependent decreases in LC3-II with unaffected ATG5 and p62/SQSTM1, suggesting impaired autophagy at least in older-aged discs [ 20 ]. Further investigation is necessary.…”

“…The present multi-color immunofluorescence for autophagy, apoptosis, and notochordal markers further identified robust autophagy in notochordal cells of non-degenerated discs and decreased autophagy but increased apoptosis in non-notochordal cells of temporary static compression-induced degenerative discs. The pro-survival effects of autophagy are well known in diverse cell types [ 49 ], including disc cells and chondrocytes [ 20 , 21 , 22 , 23 , 41 , 42 , 50 ]. In this study, the relationship between autophagy and apoptosis is unclear, although findings suggest an additional role of autophagy in maintaining the notochordal phenotype in disc NP cells by limiting apoptosis.…”

“…It is unknown whether distributed autophagy-related proteins are remnants of the notochordal phenotype [ 10 , 11 ]. Altered autophagy levels in response to diverse stress conditions, e.g., inflammation, during degeneration are also controversial [ 20 , 44 , 45 ]. Further investigation is required to understand in vivo autophagy under chronic stress.…”

“…Thus, we hypothesized that autophagy would be pivotal in maintaining disc health and homeostasis, especially for notochordal cells originally living in the avascular, low-nutrient central NP. In fact, disc cellular autophagy [ 19 , 20 ] with related intracellular signaling networks [ 21 , 22 ] has increased interest, as summarized by a review [ 23 ]. However, few studies have focused on the relationship of notochordal cell disappearance with autophagy as well as with apoptosis.…”