Zhang Yuhua, Fang Qiaojun, Wang Hongfeng, Qi Jieyu, Sun Shan, Liao Menghui, Wu Yunhao, Hu Yangnan, Jiang Pei, Cheng Cheng, Qian Xiaoyun, Tang Mingliang, Cao Wei, Xiang Shang, Zhang Chen, Yang Jianming, Gao Xia, Ying Zheng, Chai Renjie
State Key Laboratory of Bioelectronics, Department of Otolaryngology Head and Neck Surgery, Zhongda Hospital, School of Life Sciences and Technology, Advanced Institute for Life and Health, Jiangsu Province High-Tech Key Laboratory for Bio-Medical Research, Southeast University, Nanjing, Jiangsu, China.
Jiangsu Key Laboratory of Neuropsychiatric Diseases and College of Pharmaceutical Sciences, Soochow University, Suzhou, Jiangsu, China.
Autophagy. 2023 Jan;19(1):75-91. doi: 10.1080/15548627.2022.2062872. Epub 2022 Apr 26.
Aminoglycosides exhibit ototoxicity by damaging mitochondria, which in turn generate reactive oxygen species that induce hair cell death and subsequent hearing loss. It is well known that damaged mitochondria are degraded by mitophagy, an important mitochondrial quality control system that maintains mitochondrial homeostasis and ensures cell survival. However, it is unclear whether dysregulation of mitophagy contributes to aminoglycoside-induced hair cell injury. In the current study, we found that PINK1-PRKN-mediated mitophagy was impaired in neomycin-treated hair cells. Our data suggested that mitochondrial recruitment of PRKN and phagophore recognition of damaged mitochondria during mitophagy were blocked following neomycin treatment. In addition, the degradation of damaged mitochondria by lysosomes was significantly decreased as indicated by the mitophagic flux reporter mt-mKeima. Moreover, we demonstrated that neomycin disrupted mitophagy through transcriptional inhibition of expression, the key initiator of mitophagy. Moreover, we found that neomycin impaired mitophagy by inducing ATF3 expression. Importantly, treatment with a mitophagy activator could rescue neomycin-treated hair cells by increasing mitophagy, indicating that genetic modulation or drug intervention in mitophagy may have therapeutic potential for aminoglycoside-induced hearing loss. AAV: adeno-associated virus; ABR: auditory brainstem response; ATF3: activating transcription factor 3; ATOH1/MATH1: atonal bHLH transcription factor 1; BafA1: bafilomycin A; CCCP: carbonyl cyanide m-chlorophenyl hydrazone; COX4I1/COXIV: cytochrome c oxidase subunit 4I1; CTBP2/RIBEYE: C-terminal binding protein 2; DFP: deferiprone; EGFP: enhanced green fluorescent protein; FOXO3: forkhead box O3; GRIA2/GLUR2: glutamate receptor, ionotropic, AMPA2 (alpha 2); HC: hair cell; HSPD1/HSP60: heat shock protein 1 (chaperonin); IHC: inner hair cell; MAP1LC3B/LC3B: microtubule-associated protein 1 light chain 3 beta; MYO7A: myosin VIIA; OPTN: optineurin; OMM: outer mitochondrial membrane; PRKN: parkin RBR E3 ubiquitin protein ligase; PINK1: PTEN induced putative kinase 1; RT-qPCR: real-time quantitative polymerase chain reaction; TOMM20/TOM20: translocase of outer mitochondrial membrane 20; TUNEL: Terminal deoxynucleotidyl transferase (TdT) dUTP nick-end labeling; USP30: ubiquitin specific peptidase 30; XBP1: X-box binding protein 1.
氨基糖苷类药物通过损伤线粒体而表现出耳毒性，线粒体会进而产生活性氧，诱导毛细胞死亡并导致随后的听力损失。众所周知，受损的线粒体通过线粒体自噬被降解，线粒体自噬是一种重要的线粒体质量控制系统，可维持线粒体稳态并确保细胞存活。然而，尚不清楚线粒体自噬失调是否会导致氨基糖苷类药物诱导的毛细胞损伤。在本研究中，我们发现新霉素处理的毛细胞中，由PINK1-PRKN介导的线粒体自噬受损。我们的数据表明，新霉素处理后，线粒体自噬过程中PRKN向线粒体的募集以及受损线粒体的吞噬体识别被阻断。此外，线粒体自噬通量报告基因mt-mKeima显示，溶酶体对受损线粒体的降解显著减少。此外，我们证明新霉素通过转录抑制线粒体自噬的关键启动因子的表达来破坏线粒体自噬。此外，我们发现新霉素通过诱导激活转录因子3（ATF3）的表达来损害线粒体自噬。重要的是，用线粒体自噬激活剂进行治疗可以通过增加线粒体自噬来挽救新霉素处理的毛细胞，这表明对线粒体自噬进行基因调控或药物干预可能对氨基糖苷类药物诱导的听力损失具有治疗潜力。AAV：腺相关病毒；ABR：听觉脑干反应；ATF3：激活转录因子3；ATOH1/MATH1：无调性bHLH转录因子1；BafA1：巴佛洛霉素A；CCCP：羰基氰化物间氯苯腙；COX4I1/COXIV：细胞色素c氧化酶亚基4I1；CTBP2/RIBEYE：C末端结合蛋白2；DFP：去铁酮；EGFP：增强型绿色荧光蛋白；FOXO3：叉头框O3；GRIA2/GLUR2：离子型谷氨酸受体AMPA2（α2）；HC：毛细胞；HSPD1/HSP60：热休克蛋白1（伴侣蛋白）；IHC：内毛细胞；MAP1LC3B/LC3B：微管相关蛋白1轻链3β；MYO7A：肌球蛋白VIIA；OPTN：视紫质；OMM：线粒体外膜；PRKN：帕金RBR E3泛素蛋白连接酶；PINK1：PTEN诱导的假定激酶1；RT-qPCR：实时定量聚合酶链反应；TOMM20/TOM20：线粒体外膜转位酶20；TUNEL：末端脱氧核苷酸转移酶（TdT）dUTP缺口末端标记；USP30：泛素特异性肽酶30；XBP1：X盒结合蛋白1。
