UCHL1 alleviates nucleus pulposus cell senescence by promoting chaperone-mediated autophagy antagonizing autophagy-dependent ferroptosis through deubiquitination of HSPA8

Chaperone-mediated autophagy (CMA), a lysosome-dependent protein degradation pathway, plays a pivotal yet poorly understood role in cellular senescence-related degenerative diseases. Our study sheds light on a novel mechanism whereby UCHL1 plays a crucial role in mitigating nucleus pulposus cell (NPC) senescence and intervertebral disc degeneration (IVDD) by activating CMA to counteract autophagy-dependent ferroptosis. Through sequencing analysis of human samples, we identified UCHL1 as a potential factor influencing disc degeneration. Further research revealed that UCHL1 activates CMA by stabilizing HSPA8 through deubiquitination. HSPA8, in turn, recognizes and promotes the degradation of HPCAL1 via the CMA pathway by binding to its “KFERQ” motif, ultimately alleviating NPC senescence. Importantly, we demonstrated that engineered exosomes delivering <i>UCHL1</i>-overexpressing plasmids effectively alleviated NPC senescence and significantly mitigated the progression of IVDD. This finding underscores the significance of CMA-regulated ferroptosis in IVDD through UCHL1 modulation and as a promising target for improving chronic pain and IVDD progression. <b>Abbreviations:</b> AAV: adeno-associated virus; AB: Alcian Blue; ACSL4: acyl-CoA synthetase long chain family member 4; ALP: autophagy-lysosome pathway; Baf-A1: bafilomycin A1; CHX: cycloheximide; CMA: chaperone-mediated autophagy; Co-IP: co-immunoprecipitation; DUBs: deubiquitinating enzymes; eMI: endosomal microautophagy; Evs: extracellular vesicles; Exo: exosome; GPX4: glutathione peroxidase 4; H&amp;E: hematoxylin and eosin; HsNPCs: Human NPCs; IF: immunofluorescence; IHC: immunohistochemistry; IP-MS: immunoprecipitation mass spectrometry; IVDD: intervertebral disc degeneration; IVDs: intervertebral discs; LBP: low back pain; LDP: lumbar disc prolapse; MRI: magnetic resonance imaging; N/L: NH4Cl and leupeptin; NP: nucleus pulposus; NPCs: nucleus pulposus cells; PCA: principal component analysis; qRT-PCR: quantitative real-time PCR; RnBMSCs: rat bone marrow mesenchymal stem cells; RnNPCs: rat NPCs; ROS: reactive oxygen species; SA-GLB1/β-gal: senescence-associated galactosidase beta 1; SASP: senescence-associated secretory phenotype; SD: Sprague-Dawley; SO: Safranin O-Fast Green; TBHP: tert-butyl hydroperoxide; UCHL1: ubiquitin C-terminal hydrolase L1; UPS: ubiquitin-proteasome system

分子伴侣介导的自噬（Chaperone-mediated autophagy, CMA）是一类依赖溶酶体的蛋白质降解通路，在细胞衰老相关退行性疾病中发挥关键却尚未被充分阐明的作用。本研究揭示了一种全新调控机制：泛素羧基末端水解酶L1（ubiquitin C-terminal hydrolase L1, UCHL1）可通过激活CMA拮抗自噬依赖性铁死亡，从而有效缓解髓核细胞（nucleus pulposus cell, NPC）衰老与椎间盘退变（intervertebral disc degeneration, IVDD）。通过对人类样本的测序分析，我们将UCHL1鉴定为影响椎间盘退变的潜在调控因子。进一步研究发现，UCHL1通过去泛素化稳定热休克蛋白家族A成员8（heat shock protein family A member 8, HSPA8），进而激活CMA通路。HSPA8可通过结合HPCAL1的"KFERQ"基序，识别并介导其经CMA通路降解，最终延缓髓核细胞衰老。值得注意的是，我们证实递送过表达*UCHL1*质粒的工程化外泌体（exosome, Exo）可有效缓解髓核细胞衰老，并显著延缓IVDD的进展。该研究凸显了通过UCHL1调控CMA依赖的铁死亡在IVDD中的重要意义，同时也为改善慢性疼痛与延缓IVDD进展提供了极具潜力的干预靶点。**缩写：** AAV：腺相关病毒（adeno-associated virus）；AB：阿尔辛蓝（Alcian Blue）；ACSL4：长链酰基辅酶A合成酶家族成员4（acyl-CoA synthetase long chain family member 4）；ALP：自噬溶酶体通路（autophagy-lysosome pathway）；Baf-A1：巴弗洛霉素A1（bafilomycin A1）；CHX：环己酰亚胺（cycloheximide）；CMA：分子伴侣介导的自噬（chaperone-mediated autophagy）；Co-IP：免疫共沉淀（co-immunoprecipitation）；DUBs：去泛素化酶（deubiquitinating enzymes）；eMI：内体微自噬（endosomal microautophagy）；Evs：细胞外囊泡（extracellular vesicles）；Exo：外泌体（exosome）；GPX4：谷胱甘肽过氧化物酶4（glutathione peroxidase 4）；H&E：苏木精-伊红染色（hematoxylin and eosin）；HsNPCs：人髓核细胞（Human NPCs）；IF：免疫荧光（immunofluorescence）；IHC：免疫组织化学（immunohistochemistry）；IP-MS：免疫沉淀质谱法（immunoprecipitation mass spectrometry）；IVDD：椎间盘退变（intervertebral disc degeneration）；IVDs：椎间盘（intervertebral discs）；LBP：下腰痛（low back pain）；LDP：腰椎间盘突出症（lumbar disc prolapse）；MRI：磁共振成像（magnetic resonance imaging）；N/L：氯化铵与亮肽素（NH4Cl and leupeptin）；NP：髓核（nucleus pulposus）；NPCs：髓核细胞（nucleus pulposus cells）；PCA：主成分分析（principal component analysis）；qRT-PCR：实时定量聚合酶链式反应（quantitative real-time PCR）；RnBMSCs：大鼠骨髓间充质干细胞（rat bone marrow mesenchymal stem cells）；RnNPCs：大鼠髓核细胞（rat NPCs）；ROS：活性氧（reactive oxygen species）；SA-GLB1/β-gal：衰老相关β-半乳糖苷酶（senescence-associated galactosidase beta 1）；SASP：衰老相关分泌表型（senescence-associated secretory phenotype）；SD：斯普拉格-道利品系（Sprague-Dawley）；SO：番红O-固绿染色（Safranin O-Fast Green）；TBHP：叔丁基过氧化氢（tert-butyl hydroperoxide）；UCHL1：泛素羧基末端水解酶L1（ubiquitin C-terminal hydrolase L1）；UPS：泛素-蛋白酶体系统（ubiquitin-proteasome system）