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      Blebbistatin Inhibits Neomycin-Induced Apoptosis in Hair Cell-Like HEI-OC-1 Cells and in Cochlear Hair Cells

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          Abstract

          Aging, noise, and ototoxic drug-induced hair cell (HC) loss are the major causes of sensorineural hearing loss. Aminoglycoside antibiotics are commonly used in the clinic, but these often have ototoxic side effects due to the accumulation of oxygen-free radicals and the subsequent induction of HC apoptosis. Blebbistatin is a myosin II inhibitor that regulates microtubule assembly and myosin–actin interactions, and most research has focused on its ability to modulate cardiac or urinary bladder contractility. By regulating the cytoskeletal structure and reducing the accumulation of reactive oxygen species (ROS), blebbistatin can prevent apoptosis in many different types of cells. However, there are no reports on the effect of blebbistatin in HC apoptosis. In this study, we found that the presence of blebbistatin significantly inhibited neomycin-induced apoptosis in HC-like HEI-OC-1 cells. We also found that blebbistatin treatment significantly increased the mitochondrial membrane potential (MMP), decreased ROS accumulation, and inhibited pro-apoptotic gene expression in both HC-like HEI-OC-1 cells and explant-cultured cochlear HCs after neomycin exposure. Meanwhile, blebbistatin can protect the synaptic connections between HCs and cochlear spiral ganglion neurons. This study showed that blebbistatin could maintain mitochondrial function and reduce the ROS level and thus could maintain the viability of HCs after neomycin exposure and the neural function in the inner ear, suggesting that blebbistatin has potential clinic application in protecting against ototoxic drug-induced HC loss.

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          Most cited references 35

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          Mechanism of blebbistatin inhibition of myosin II.

          Blebbistatin is a recently discovered small molecule inhibitor showing high affinity and selectivity toward myosin II. Here we report a detailed investigation of its mechanism of inhibition. Blebbistatin does not compete with nucleotide binding to the skeletal muscle myosin subfragment-1. The inhibitor preferentially binds to the ATPase intermediate with ADP and phosphate bound at the active site, and it slows down phosphate release. Blebbistatin interferes neither with binding of myosin to actin nor with ATP-induced actomyosin dissociation. Instead, it blocks the myosin heads in a products complex with low actin affinity. Blind docking molecular simulations indicate that the productive blebbistatin-binding site of the myosin head is within the aqueous cavity between the nucleotide pocket and the cleft of the actin-binding interface. The property that blebbistatin blocks myosin II in an actin-detached state makes the compound useful both in muscle physiology and in exploring the cellular function of cytoplasmic myosin II isoforms, whereas the stabilization of a specific myosin intermediate confers a great potential in structural studies.
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            The aminoglycoside antibiotic dihydrostreptomycin rapidly enters mouse outer hair cells through the mechano-electrical transducer channels.

            The most serious side-effect of the widely used aminoglycoside antibiotics is irreversible intracellular damage to the auditory and vestibular hair cells of the inner ear. The mechanism of entry into the hair cells has not been unequivocally resolved. Here we report that extracellular dihydrostreptomycin not only blocks the mechano-electrical transducer channels of mouse outer hair cells at negative membrane potentials, as previously shown, but also enters the cells through these channels, which are located in the cells' mechanosensory hair bundles. The voltage-dependent blocking kinetics indicate an open-channel block mechanism, which can be well described by a two barrier-one binding site model, quantifying the antibiotic's block of the channel as well as its permeation in terms of the associated rate constants. The results identify the open transducer channels as the main route for aminoglycoside entry. Intracellularly applied dihydrostreptomycin also blocks the transducer channels, but at positive membrane potentials. However, the potency of the block was two orders of magnitude lower than that due to extracellular dihydrostreptomycin. Extracellular Ca2+ increases the free energy of the barrier nearest the extracellular side and of the binding site for dihydrostreptomycin. This reduces both the entry of dihydrostreptomycin into the channel and the channel's affinity for the drug. In vivo, where the extracellular Ca2+ concentration in the endolymph surrounding the hair bundles is < 100 microM, we predict that some 9000 dihydrostreptomycin molecules per second enter each hair cell at therapeutic drug concentrations.
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              Autophagy protects auditory hair cells against neomycin-induced damage

              ABSTRACT Aminoglycosides are toxic to sensory hair cells (HCs). Macroautophagy/autophagy is an essential and highly conserved self-digestion pathway that plays important roles in the maintenance of cellular function and viability under stress. However, the role of autophagy in aminoglycoside-induced HC injury is unknown. Here, we first found that autophagy activity was significantly increased, including enhanced autophagosome-lysosome fusion, in both cochlear HCs and HEI-OC-1 cells after neomycin or gentamicin injury, suggesting that autophagy might be correlated with aminoglycoside-induced cell death. We then used rapamycin, an autophagy activator, to increase the autophagy activity and found that the ROS levels, apoptosis, and cell death were significantly decreased after neomycin or gentamicin injury. In contrast, treatment with the autophagy inhibitor 3-methyladenine (3-MA) or knockdown of autophagy-related (ATG) proteins resulted in reduced autophagy activity and significantly increased ROS levels, apoptosis, and cell death after neomycin or gentamicin injury. Finally, after neomycin injury, the antioxidant N-acetylcysteine could successfully prevent the increased apoptosis and HC loss induced by 3-MA treatment or ATG knockdown, suggesting that autophagy protects against neomycin-induced HC damage by inhibiting oxidative stress. We also found that the dysfunctional mitochondria were not eliminated by selective autophagy (mitophagy) in HEI-OC-1 cells after neomycin treatment, suggesting that autophagy might not directly target the damaged mitochondria for degradation. This study demonstrates that moderate ROS levels can promote autophagy to recycle damaged cellular constituents and maintain cellular homeostasis, while the induction of autophagy can inhibit apoptosis and protect the HCs by suppressing ROS accumulation after aminoglycoside injury.
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                Author and article information

                Contributors
                Journal
                Front Cell Neurosci
                Front Cell Neurosci
                Front. Cell. Neurosci.
                Frontiers in Cellular Neuroscience
                Frontiers Media S.A.
                1662-5102
                05 February 2020
                2019
                : 13
                Affiliations
                1Department of Otolaryngology, Affiliated People’s Hospital of Jiangsu University , Zhenjiang, China
                2Department of Otolaryngology Head and Neck Surgery, Nanjing Drum Tower Hospital Clinical College of Nanjing Medical University, Jiangsu Provincial Key Medical Discipline (Laboratory) , Nanjing, China
                3Research Institute of Otolaryngology , Nanjing, China
                4Department of Otolaryngology, Head and Neck Surgery, XiangYa School of Medicine, Central South University , Changsha, China
                5MOE Key Laboratory for Developmental Genes and Human Disease, Institute of Life Sciences, Southeast University , Nanjing, China
                6Department of Otolaryngology Head and Neck Surgery, Shanghai Jiao Tong University Affiliated Sixth People’s Hospital , Shanghai, China
                7Co-Innovation Center of Neuroregeneration, Nantong University , Nantong, China
                8Institute for Stem Cell and Regeneration, Chinese Academy of Science , Beijing, China
                9Beijing Key Laboratory of Neural Regeneration and Repair, Capital Medical University , Beijing, China
                10Jiangsu Province High-Tech Key Laboratory for Bio-Medical Research, Southeast University , Nanjing, China
                Author notes

                Edited by: Zhang Pengyue, Yunnan University of Traditional Chinese Medicine, China

                Reviewed by: Wenwen Liu, Shandong University, China; Bingjin Li, Jilin University, China

                These authors have contributed equally to this work

                Article
                10.3389/fncel.2019.00590
                7025583
                Copyright © 2020 Gao, Cheng, Wang, Jiang, Zhang, Wang, Wu, Zeng, Wang, Gao, Ma and Chai.

                This is an open-access article distributed under the terms of the Creative Commons Attribution License (CC BY). The use, distribution or reproduction in other forums is permitted, provided the original author(s) and the copyright owner(s) are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. No use, distribution or reproduction is permitted which does not comply with these terms.

                Page count
                Figures: 8, Tables: 1, Equations: 0, References: 43, Pages: 12, Words: 6409
                Funding
                Funded by: Innovative Research Group Project of the National Natural Science Foundation of China 10.13039/100014718
                Award ID: 81970884, 81900941, 81900944, 81622013, 81970882, 81570919, 81870721, 81771019, 81700913, 81670928, 81570921
                Funded by: Natural Science Foundation of Jiangsu Province 10.13039/501100004608
                Funded by: K. C. Wong Education Foundation 10.13039/501100012692
                Categories
                Cellular Neuroscience
                Original Research

                Neurosciences

                aminoglycoside, hair cell, blebbistatin, apoptosis, ros, hearing, synaptic plasticity

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