• Helen E Gruber, Gretchen L Hoelscher, Jane A Ingram, Synthia Bethea, and Edward N Hanley.
• From the Department of Orthopaedic Surgery, Carolinas HealthCare System, Charlotte, NC.
• Spine. 2015 Jun 1; 40 (11): 773782773-82.

Study DesignAutophagy-related gene expression and ultrastructural features of autophagy were studied in human discs.ObjectiveTo obtain molecular/morphological data on autophagy in human disc degeneration and cultured human annulus cells exposed to proinflammatory cytokines.Summary Of Background DataAutophagy is an important process by which cytoplasm and organelles are degraded; this adaptive response to sublethal stresses (such as nutrient deprivation present in disc degeneration) supplies needed metabolites. Little is known about autophagic processes during disc degeneration.MethodsHuman disc specimens were obtained after institutional review board approval. Annulus mRNA was analyzed to determine autophagy-related gene expression levels. Immunolocalization and ultrastructural studies for p62, ATG3, ATG4B, ATG4C, ATG7, L3A, ULK-2, and beclin were conducted. In vitro experiments used IL-1β- or TNF-α-treated human annulus cells to test for autophagy-related gene expression.ResultsMore degenerated versus healthier discs showed significantly greater upregulation of well-recognized autophagy-related genes (P ≤ 0.028): beclin 1 (upregulated 1.6-fold); ATG8 (LC3) (upregulated 2.0-fold); ATG12 (upregulated 4.0-fold); presenilin 1 (upregulated 1.6-fold); cathepsin B (upregulated 4.5-fold). p62 was localized, and ultrastructure showed autophagic vacuolization and autophagosomes with complex, redundant whorls of membrane-derived material. In vitro, proinflammatory cytokines significantly upregulated autophagy-related genes (P ≤ 0.04): DRAM1 (6.24-fold); p62 (4.98-fold); PIM-2 oncogene, a positive regulator of autophagy (3-fold); WIPI49 (linked to starvation-induced autophagy) (upregulated 2.3-fold).ConclusionData provide initial molecular and morphological evidence for the presence of autophagy in the degenerating human annulus. In vivo gene analyses showed greater autophagy-related gene expression in more degenerated than healthier discs. In vitro data suggested a mechanism implicating a role of TNF-α and IL-1β in disc autophagy. Findings suggest the importance of future work to investigate the relationship of autophagy to apoptosis, cell death, cell senescence, and mitochondrial dysfunction in the aging and degenerating disc.Level Of EvidenceN/A.

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