inhibition may induce in a calcium-dependent manner.
CMA defects are linked to cellular senescence and .
PLCG1 was identified as a key mediator affecting calcium levels in cells.
Accumulation of PLCG1 due to CMA blockage leads to calcium overload.
Evidence from human specimens shows reduced LAMP2A or increased PLCG1 correlates with disc senescence.
In a rat model, TNF-induced disc degeneration was inhibited by modulating PLCG1 levels.
Simplified
Defects in (CMA) are associated with , but the mechanism remains poorly understood. Here, we found that CMA inhibition induced cellular senescence in a calcium-dependent manner and identified its role in TNF-induced senescence of nucleus pulposus cells (NPC) and . Based on structural and functional proteomic screens, PLCG1 (phospholipase C gamma 1) was predicted as a potential substrate for CMA deficiency to affect calcium homeostasis. We further confirmed that PLCG1 was a key mediator of CMA in the regulation of intracellular calcium flux. Aberrant accumulation of PLCG1 caused by CMA blockage resulted in calcium overload, thereby inducing NPC senescence. Immunoassays on human specimens showed that reduced LAMP2A, the rate-limiting protein of CMA, or increased PLCG1 was associated with disc senescence, and the TNF-induced disc degeneration in rats was inhibited by overexpression ofor knockdown of. Because CMA dysregulation, calcium overload, and cellular senescence are common features of disc degeneration and other age-related degenerative diseases, the discovery of actionable molecular targets that can link these perturbations may have therapeutic value.ATRA: all-trans-retinoic acid; BrdU: bromodeoxyuridine; CDKN1A/p21: cyclin dependent kinase inhibitor 1A; CDKN2A/p16-INK4A: cyclin dependent kinase inhibitor 2A; CMA: chaperone-mediated autophagy; DHI: disc height index; ER: endoplasmic reticulum; IP: immunoprecipitation; IP3: inositol 1,4,5-trisphosphate; ITPR/IP3R: inositol 1,4,5-trisphosphate receptor; IVD: intervertebral disc; IVDD: intervertebral disc degeneration; KD: knockdown; KO: knockout; Leu: leupeptin; MRI: magnetic resonance imaging; MS: mass spectrometry; N/L: NHCl and leupeptin; NP: nucleus pulposus; NPC: nucleus pulposus cells; PI: protease inhibitors; PLC: phospholipase C; PLCG1: phospholipase C gamma 1; ROS: reactive oxygen species; RT-qPCR: real-time quantitative reverse transcription PCR; SA-GLB1/β-gal: senescence-associated galactosidase beta 1; SASP: senescence-associated secretory phenotype; STV: starvation; TMT: tandem mass tag; TNF: tumor necrosis factor; TP53: tumor protein p53; UPS: ubiquitin-proteasome system. Lamp2a Plcg1Abbreviation: 4
Key numbers
78
Human disc samples analyzed
Patients aged 39–72 years undergoing discectomy for disc herniation or spinal stenosis.
20 ng/mL
Significant increase in senescence markers
TNF treatment level used to assess senescence induction in NPC.
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