USP39 inhibits MLKL phosphorylation and deubiquitination to suppress necroptosis of nucleus pulposus cells and attenuate intervertebral disc degeneration

Intervertebral disc degeneration (IVDD) is a leading cause of chronic low back pain. Programmed cell death, particularly necroptosis, contributes to nucleus pulposus (NP) cell loss. Mixed lineage kinase domain-like protein (MLKL) phosphorylation plays a critical role in necroptotic execution, but its upstream regulation in IVDD remains poorly defined. We analyzed human and mouse degenerative disc tissues, as well as TNF-α/Smac mimetic/Z-VAD-FMK (TSZ)-treated NP cells, to assess MLKL phosphorylation. MLKL knockdown in human NP cells and conditional knockout (CKO) in mice were performed to determine its functional role. Immunoprecipitation coupled with mass spectrometry identified potential MLKL-binding proteins. Functional assays with USP39 knockdown/overexpression, together with in vitro and in vivo IVDD models, were conducted to explore regulatory mechanisms. MLKL phosphorylation was markedly elevated in human IVDD tissues, LSI-induced mouse discs, and TSZ-stimulated NP cells. Genetic knockdown or conditional deletion of Mlkl significantly preserved extracellular matrix integrity and delayed degeneration. USP39 was identified as a novel MLKL-interacting deubiquitinase. USP39 expression was reduced in IVDD, and its overexpression inhibited MLKL ubiquitination and phosphorylation, alleviating NP cell degeneration. In vivo, AAV-mediated Usp39 delivery attenuated disc degeneration and suppressed MLKL activation. Our study reveals that MLKL phosphorylation drives necroptosis in IVDD and identifies USP39 as a critical upstream regulator that deubiquitinates MLKL. Targeting the USP39-MLKL axis provides a promising therapeutic strategy for delaying IVDD progression. This study reveals that USP39 inhibits MLKL phosphorylation through deubiquitination, thereby suppressing necroptosis of nucleus pulposus cells and alleviating IVDD. Targeting the USP39–MLKL axis provides a potential therapeutic strategy to preserve NP cell viability and slow the progression of IVDD, offering new insight for translational interventions in chronic low back pain. This study investigates the role of USP39 in regulating MLKL-mediated necroptosis in nucleus pulposus (NP) cells during intervertebral disc degeneration (IVDD). MLKL phosphorylation is significantly elevated in degenerative tissues and TSZ-stimulated NP cells. USP39 is identified as a deubiquitinase that deubiquitinates MLKL, reducing its phosphorylation and preventing necroptosis. This protective mechanism delays extracellular matrix (ECM) degradation by maintaining NP cell viability, as seen in both in vitro and in vivo models. Genetic knockdown of MLKL or USP39 overexpression significantly alleviates degeneration, offering potential therapeutic strategies for IVDD progression by targeting the USP39-MLKL axis.