The USP5-MATN3 axis in nucleus pulposus cell senescence: A Deubiquitination mechanism protecting against intervertebral disc degeneration.

MGST1 Suppresses Ferroptosis in Nucleus Pulposus Cells and Attenuates Intervertebral Disc Degeneration by Regulating GPX4.

MGST1 exhibits compensatory upregulation during the progression of intervertebral disc degeneration and delays its progression by inhibiting ferroptosis in nucleus pulposus cells, a novel mechanism by which MGST1 regulates ferroptosis in IDD and provides a potential molecular target for the prevention and treatment of IDD.

Impaired Chaperone‐Mediated Autophagy Accelerates Intervertebral Disc Degeneration by Inducing MIDN Accumulation to Target TSC2 for Proteasomal Degradation

ABSTRACT Intervertebral disc degeneration (IDD) is a leading cause of low back pain with incompletely understood mechanisms. Although autophagy dysfunction is a documented contributor to IDD, the precise pathobiological role of chaperone‐mediated autophagy (CMA) remains poorly understood. Here, we demonstrate that CMA activity is downregulated in nucleus pulposus cells (NPCs) from IDD patients and IL‐1β‐induced rat intervertebral disc cell models, causing cytoplasmic accumulation of a novel CMA substrate, Midnolin (MIDN). Accumulated MIDN bypasses the ubiquitin‐proteasome system and directly binds to Tuberous Sclerosis Complex 2 (TSC2), mediating its degradation. TSC2 loss relieves mechanistic target of rapamycin complex 1 (mTORC1) inhibition, resulting in mTORC1 hyperactivation, which drives cellular senescence, senescence‐associated secretory phenotype (SASP), and extracellular matrix (ECM) degradation in NPCs. In vitro and in a rat caudal needle puncture model, MIDN knockdown (shRNA), CMA activation (LAMP2A overexpression), or mTORC1 inhibition (Rapamycin) significantly attenuated IL‐1β or MIDN overexpression‐induced senescence and disc degeneration. Our findings reveal an “Impaired CMA–MIDN accumulation–TSC2 degradation–mTORC1 activation” axis central to IDD pathogenesis, offering potential therapeutic targets.

MANF safeguards mitochondria-associated endoplasmic reticulum membrane integrity in nucleus pulposus-derived mesenchymal stem cells to maintain homeostasis of the intervertebral disc.

BACKGROUND Intervertebral disc (IVD) degeneration (IDD) is a leading cause of low back pain, with limited treatment options. The degenerative disc's harsh microenvironment promotes nucleus pulposus-derived mesenchymal stem cells (NP-MSCs) death and hinders self repair. Mesencephalic astrocyte-derived neurotrophic factor (MANF), an atypical neurotrophic factor, has protective effects in degenerative diseases. However, its role in IDD is unclear. METHODS Assessment of MANF expression was conducted in both human nucleus pulposus tissues and a rat IVD puncture model. An in vitro model of degeneration was established by acid treatment of NP-MSCs, and the functional role of MANF was explored through its knockdown and overexpression. RNA sequencing was employed to identify downstream targets. The therapeutic potential of MANF-overexpressing NP-MSCs was evaluated in a rat puncture model. RESULTS MANF expression was markedly downregulated in degenerated IVD tissues from both human patients and rat models. Correspondingly, in vitro experiments demonstrated that MANF knockdown exacerbated, while its overexpression mitigated, acid-induced apoptosis of NP-MSCs. Mechanistically, MANF attenuated mitochondrial dysfunction and ER stress of NP-MSCs under acidic conditions by maintaining MAM integrity, as demonstrated by the complete abolition of this protection upon treatment with the MAM uncoupler FATE1. Transcriptomic analysis and subsequent validation identified receptor expression-enhancing protein 1 (REEP1) as a critical downstream effector through which MANF safeguards MAM integrity. We further elucidated that MANF upregulates REEP1 expression by directly inhibiting miR-33b-5p. In vivo, transplantation of MANF-overexpressing NP-MSCs effectively attenuated IDD in a rat model. CONCLUSION MANF protected NP-MSCs from acidosis by sustaining MAM integrity via the MiR-33b-5p/REEP1 axis. These findings reveal MANF's mechanism and therapeutic potential for IDD.

FOXO3 Counteracts Oxidative Stress-Induced Nucleus Pulposus Cell Senescence via BNIP3-Mediated Mitophagy in Intervertebral Disc Degeneration.

Oxidative stress and mitochondrial reactive oxygen species (ROS) accumulation are central drivers of nucleus pulposus (NP) cell senescence and intervertebral disc degeneration (IVDD), yet the transcriptional programs that maintain mitochondrial redox homeostasis in NP cells remain poorly defined. In this study, we investigated the role of forkhead box O3 (FOXO3) in regulating NP cell senescence and mitophagy during IVDD. We found that FOXO3 expression was significantly reduced in degenerated disc tissues and in senescent NP cells. In vitro, FOXO3 overexpression markedly alleviated H2O2-induced senescence, as shown by reduced SA-β-gal positivity, downregulation of p16 and senescence-associated catabolic factors, and partial restoration of extracellular matrix-related proteins. Transcriptomic profiling revealed that FOXO3 suppressed senescence-, NF-κB-, and matrix degradation-associated pathways while activating autophagy-related programs. Mechanistically, FOXO3 enhanced autophagic flux and promoted BNIP3-dependent mitophagy, accompanied by improved mitochondrial ultrastructure, preservation of mitochondrial membrane potential, and reduced mitochondrial reactive oxygen species accumulation. Importantly, BNIP3 knockdown partially abolished the anti-senescent effects of FOXO3, supporting BNIP3-mediated mitophagy as a key downstream mechanism. In vivo, adeno-associated virus-mediated FOXO3 overexpression attenuated puncture-induced disc degeneration, reduced p16 and MMP13 expression, and restored aggrecan and collagen II levels. Collectively, these findings identify FOXO3 as a critical regulator of mitochondrial quality control and NP cell senescence, and suggest that targeting the FOXO3-BNIP3-mitophagy axis may represent a promising therapeutic strategy for IVDD.

Integrated bioinformatics and experimental validation reveal that kaempferol ameliorates intervertebral disc degeneration via dual anti-inflammatory and anti-aging pathways.

Inhibition of ETS1/DRP1 axis attenuates intervertebral disc degeneration by suppressing excessive mitochondrial fission.

Intervertebral disc degeneration (IVDD) is a predominant contributor to low back pain, characterized by nucleus pulposus cell (NPC) senescence, extracellular matrix (ECM) metabolic dysfunction, and chronic inflammation. Excessive mitochondrial fission contributes to IVDD, yet the underlying regulatory mechanisms remain unclear. Herein, we identified ETS proto-oncogene 1 (ETS1) as a critical regulator of mitochondrial fission in human NPCs. ETS1 was upregulated in severe human IVDD and correlated with disc degeneration severity and NPC senescence. Mechanistically, inflammatory cytokines induced ETS1 upregulation, which directly bound to the dynamin 1-like (DNM1L, encoding DRP1) promoter and activated its transcription. Increased DRP1 triggered excessive mitochondrial fission, leading to reactive oxygen species accumulation, NPC senescence, and ECM catabolism. Inhibition of ETS1 via AAV5-mediated RNA interference or targeting DRP1 with CRISPR/dCas9-KRAB system or Mdivi-1 alleviated mitochondrial dysfunction, cellular senescence, ECM degradation, and attenuated IVDD progression. Collectively, our findings revealed the ETS1/DRP1 axis as a novel pathogenic mechanism and a potential therapeutic target in IVDD.