Abstract：
Ototoxicity is a major side effect of aminoglycosides, which can cause irreversible hearing loss. Previous studies on aminoglycoside-induced ototoxicity have primarily focused on the loss of sensory hair cells. Recent investigations have revealed that aminoglycosides can also lead to the loss of ribbon synapses in inner hair cells (IHCs). However, the functional implications of ribbon synapse loss and the underlying mechanisms remain unclear. In this study, we intraperitoneally injected C57BL/6 J mice with 300 mg/kg gentamicin once daily for 3, 10, and 20 days. Then, we performed immunofluorescence staining, patch-clamp recording, proteomics analysis and western blotting to characterize the changes in ribbon synapses in IHCs and the associated mechanisms. After gentamicin treatment, the auditory brainstem response (ABR) threshold was elevated, and the ABR wave I amplitude was decreased. We also observed loss of ribbon synapses in IHCs. Interestingly, ribbon synapse loss occurred on both the modiolar and pillar sides of IHCs. Whole-cell patch-clamp recordings in IHCs revealed a reduction in the calcium current amplitude, along with a shifted half-activation voltage and altered calcium voltage dependency. Moreover, exocytosis of IHCs was reduced, consistent with the reduction in the ABR wave I amplitude. Through proteomic analysis, western blotting, and immunofluorescence staining, we found that gentamicin treatment resulted in downregulation of myosin VI, a protein crucial for synaptic vesicle recycling and replenishment in IHCs. Furthermore, we evaluated the kinetics of endocytosis and found a significant reduction in IHC exocytosis, possibly reflecting the impact of myosin VI downregulation on synaptic vesicle recycling. In summary, our findings demonstrate that gentamicin treatment leads to synaptic dysfunction in IHCs, highlighting the important role of myosin VI downregulation in gentamicin-induced synaptic damage.
摘要：
耳毒性是氨基糖苷类的主要副作用，这可能会导致不可逆转的听力损失。先前对氨基糖苷类诱导的耳毒性的研究主要集中在感觉毛细胞的损失上。最近的研究表明，氨基糖苷还可以导致内毛细胞（IHC）中带状突触的丧失。然而,带状突触丢失的功能意义和潜在机制尚不清楚.在这项研究中,我们对C57BL/6J小鼠腹腔注射庆大霉素300mg/kg，每天一次，连续3、10和20天。然后，我们进行了免疫荧光染色，膜片钳记录，蛋白质组学分析和蛋白质印迹来表征IHC中带状突触的变化及其相关机制。庆大霉素治疗后,听觉脑干反应（ABR）阈值升高，ABR波I振幅降低。我们还观察到IHC中带状突触的丢失。有趣的是,带状突触丢失发生在IHC的牙体侧和柱侧。IHC中的全细胞膜片钳记录显示钙电流幅度降低，伴随着半激活电压的偏移和钙电压依赖性的改变。此外，IHC的胞吐减少，与ABR波I振幅的减小一致。通过蛋白质组学分析,西方印迹,免疫荧光染色，我们发现庆大霉素治疗导致肌球蛋白VI的下调，对于IHC中的突触小泡再循环和补充至关重要的蛋白质。此外，我们评估了内吞作用的动力学，发现IHC胞吐作用显着降低，可能反映了肌球蛋白VI下调对突触小泡再循环的影响。总之,我们的发现表明庆大霉素治疗会导致IHC的突触功能障碍，强调肌球蛋白VI下调在庆大霉素诱导的突触损伤中的重要作用。
