Li Xinxin, Wang Jing, Shi Lin, Wang Liang
Department of Otolaryngology, The First Affiliated Hospital of Dalian Medical University, No. 222 Zhongshan Road, Dalian, 116011, China.
Stem Cell Clinical Research Center, National Joint Engineering Laboratory, Regenerative Medicine Center, The First Affiliated Hospital of Dalian Medical University, Dalian, 116012, China.
Mol Neurobiol. 2025 Jul 14. doi: 10.1007/s12035-025-05178-9.
Sensorineural hearing loss (SNHL) has a high degree of genetic heterogeneity, with numerous mutated genes that contribute to deafness. GIPC3 gene is one of the mutated genes that can cause congenital hearing loss, which has been identified in recent years; however, the exact mechanism behind this condition remains unclear. Mitophagy is the process of selectively encapsulating and lysing damaged or dysfunctional mitochondria in order to prevent the accumulation of damaged mitochondria from damaging the cells and is of great importance in the maintenance of homeostasis in the inner ear. This paper aims to investigate the potential mechanism of sensorineural hearing loss brought on by Gipc3 mutations by observing the impact of Gipc3 expression on mitochondrial metabolism and autophagy in inner ear hair cells. In this study, The House Ear Institute Organ of Corti 1(HEI-OC1) cells and cochlear explants were cultured to change the expression level of Gipc3 by transfection, and the knockdown efficiency was examined by quantitative polymerase chain reaction (qPCR) and Western blot. Knockdown of Gipc3 inhibited cell viability and its proliferation ability. When tert-butyl hydroperoxide (t-BHP) was used to induce oxidative stress injury and knockdown of Gipc3, inner ear hair cells had a weakened ability to resist oxidative stress injury, mitochondrial metabolism was altered, and there was an accumulation of reactive oxygen species (ROS) and a reduction of mitochondrial membrane potential. Immunofluorescence and Western blot techniques demonstrated autophagy abnormalities in the mitophagy-related proteins LC3B and p62. Early endosome-dependent mitophagy is mediated by a PH domain and leucine zipper motif 1 (APPL1), and mitophagy is hampered by APPL1 deletion. We discovered that there is probably co-localization between Gipc3 and APPL1 by confocal microscopy imaging and that their trends show a positive association. In summary, Gipc3 mutations may lead to decreased mitochondrial function by inhibiting the APPL1-mediated mitophagy process. This may lead to a reduction in oxidative metabolism in hair cells, which is one potential mechanism via which Gipc3 mutations suppress mitophagy.
感音神经性听力损失（SNHL）具有高度的遗传异质性，有众多导致耳聋的突变基因。GIPC3基因是近年来已确定的可导致先天性听力损失的突变基因之一；然而，这种情况背后的确切机制仍不清楚。线粒体自噬是选择性包裹和裂解受损或功能失调的线粒体的过程，以防止受损线粒体的积累对细胞造成损害，并且在内耳稳态的维持中具有重要意义。本文旨在通过观察Gipc3表达对内耳毛细胞线粒体代谢和自噬的影响，研究Gipc3突变导致感音神经性听力损失的潜在机制。在本研究中，培养了House Ear Institute Corti 1（HEI-OC1）细胞和耳蜗外植体，通过转染改变Gipc3的表达水平，并通过定量聚合酶链反应（qPCR）和蛋白质免疫印迹法检测敲低效率。敲低Gipc3会抑制细胞活力及其增殖能力。当使用叔丁基过氧化氢（t-BHP）诱导氧化应激损伤并敲低Gipc3时，内耳毛细胞抵抗氧化应激损伤的能力减弱，线粒体代谢发生改变，活性氧（ROS）积累且线粒体膜电位降低。免疫荧光和蛋白质免疫印迹技术显示线粒体自噬相关蛋白LC3B和p62存在自噬异常。早期内体依赖性线粒体自噬由含PH结构域和亮氨酸拉链基序1（APPL1）介导，APPL1缺失会阻碍线粒体自噬。我们通过共聚焦显微镜成像发现Gipc3和APPL1之间可能存在共定位，并且它们的趋势呈正相关。总之，Gipc3突变可能通过抑制APPL1介导的线粒体自噬过程导致线粒体功能下降。这可能导致毛细胞氧化代谢减少，这是Gipc3突变抑制线粒体自噬的一种潜在机制。
