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Regulation of autophagy

a promising therapeutic target for the treatment of hearing loss

Fu, Xiaolonga; Chai, Renjiea , b , c , d , e ,*

doi: 10.1097/JBR.0000000000000031
Review Articles
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Autophagy, a ubiquitous cellular biological behavior that features a lysosome-dependent degradation pathway, is an important mechanism for cellular self-protection in eukaryotes. Autophagy plays essential roles in cell survival, renewal, material reuse and the maintenance of homeostasis. This paper reviews recent advances in understanding the physiological function of autophagy and its possible roles in auditory diseases. We focused our review on original publications on animal models, drug models, and molecular mechanisms of hearing impairment involved in the dysregulation of autophagy. As research on the mechanisms of autophagy has deepened, it has become obvious that autophagy plays essential roles not only in cell survival, but the occurrence and development of a variety of auditory-related disorder, including aminoglycoside-induced hearing loss, age-related hearing loss, and noise-induced hearing loss. While clinical treatment of such conditions via regulation of the development of autophagy is a novel idea, more time is needed to fully elucidate the specific regulatory pathways and modes of autophagy in auditory diseases. The continued study of the mechanisms and regulation of autophagy in auditory diseases will be of great significance for the future treatment and prevention of these conditions.

aKey Laboratory for Developmental Genes and Human Disease, Ministry of Education, Institute of Life Sciences, Southeast University, Nanjing, Jiangsu Province

bBeijing Key Laboratory of Neural Regeneration and Repair, Capital Medical University, Beijing

cCo-Innovation Center of Neuroregeneration, Nantong University, Nantong, Jiangsu Province

dInstitute for Stem Cell and Regeneration, Chinese Academy of Science, Beijing

eJiangsu Province High-Tech Key Laboratory for Bio-Medical Research, Southeast University, Nanjing, Jiangsu Province, China

Corresponding author: Renjie Chai, Co-Innovation Center of Neuroregeneration, Key Laboratory for Developmental Genes and Human Disease, Ministry of Education, Institute of Life Sciences, Southeast University, Nanjing, Jiangsu Province, China. E-mail: renjiec@seu.edu.cn

Received January 3, 2019

Accepted March 22, 2019

This is an open access article distributed under the terms of the Creative Commons Attribution-Non Commercial-No Derivatives License 4.0 (CCBY-NC-ND), where it is permissible to download and share the work provided it is properly cited. The work cannot be changed in any way or used commercially without permission from the journal. http://creativecommons.org/licenses/by-nc-nd/4.0

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Overview of the autophagy pathway

Autophagy is a relatively conservative and very important metabolic pathway in eukaryotic organisms that plays essential roles in the transport of intracellular substances and maintenance of the stability of the internal environment. [1,2] Three main forms of autophagy are exist: macroautophagy, microautophagy, and chaperone-mediated autophagy. [3] This review will focus on macroautophagy (hereinafter referred to as autophagy), due to that it is the main regulatory degradation mechanism used by eukaryotic cells to degrade longevity proteins and organelles. [4,5] Autophagy is considered to be an intracellular system whereby cytoplasmic components are delivered and subsequently, degraded within the lysosome. [6,7] Formation of a sequestering structure called isolation membrane is the initiation of macroautophagy. [3,8] The autophagosome is a closed double-membrane-bound structure matured by the phagophore. Then, the autophagosome is fused with a lysosome. Thereafter, the lysosome hydrolases degrade the inner membrane of the autophagosome and the substances contained in the autophagosome (Fig. 1). Autophagy is usually induced by starvation and other factors. Basic autophagy is of great significance for the quality control and homeostasis of the cytoplasmic components of postmitotic neurons, hepatocytes, cardiomyocytes, and other cells.

Figure 1

Figure 1

To perform homeostasis functions, autophagy usually occurs at a low basic level in almost all cells. Autophagy is rapidly up-regulated if cells need to produce intracellular nutrients and energy during starvation, viral infection, and exercise. When cells are ready to undergo structural remodeling during development, autophagy is also up-regulated. For example, in the case of oxidative stress, infection or accumulation of protein aggregation, the function of autophagy is to remove its own destructive cytoplasmic components. In addition, hormonal factors, nutritional status, oxygen concentration, temperature and cell density are also important clues in the control of autophagy. In recent years, the comprehensive study of autophagy mechanism has focused on the molecular cascade regulating autophagy. [9–11]

The target of rapamycin (TOR) kinase is one of the major regulators of autophagy. TOR can shut off autophagy in the presence of growth factors and abundant nutrients. The class I phosphoinositide 3-kinase/protein kinase B signaling molecules repress autophagy in response to insulin-like and other growth factor signals when link receptor tyrosine kinases to TOR activation. [12] Other regulatory molecules that control autophagy include the eukaryotic initiation factor 2α, which responds to endoplasmic reticulum stress, double-stranded RNA, and nutrient starvation; the tumor suppressor protein, p53; c-Jun-N-terminal kinase; GTPases; death-associated protein kinases; the stress-activated kinase; the inositol-trisphosphate receptor; ceramide; the endoplasmic reticulum-membrane-associated protein, Ire-1; ceramide; and calcium. [13–16]

Autophagy can remove abnormal cytoplasmic proteins and digest damaged and redundant organelles. When autophagy is disturbed, it can lead to the accumulation of abnormal proteins and organelles, disorder of normal cell growth mechanisms, and many acute and chronic diseases. Some of these, which include metabolic diseases, neurodegenerative diseases, tumors and infectious diseases, can lead to autophagy disorders. While autophagy is inhibited at different stages of the development of certain diseases, thus aggravating the disease, induced autophagy may prolong life span. Strategies to upregulate or inhibit autophagy as part of the treatment of some diseases have attracted much attention. In recent years, the regulation of autophagy has been shown to have broad research and practical potential in the treatment of hearing-related diseases. This review describes recent articles on the role of autophagy in the pathogenesis of auditory diseases, and offers new ideas for the clinical treatment of auditory diseases in the future.

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Autophagy and ototoxic drug-induced hair cells

Infectious diseases caused by many bacteria can be treated with aminoglycoside antibiotics. However, these aminoglycosides can cause many side effects in clinic particularly ototoxicity, which can result in vestibular disorders, tinnitus, and hearing loss, aminoglycoside antibiotics is limited. [17] Aminoglycoside ototoxicity is typically associated with bilateral sensorineural hearing loss. The sensory hair cells (HCs) are unable to regenerate once damaged by aminoglycoside-induced cytotoxicity.

Ototoxic drug-induced HC damage is one of the main reasons for hearing loss. Although recent studies have shown that mice have a weak ability to proliferate HCs before and after birth, adult mice have completely lost their ability to regenerate HCs to reduce the damage to hearing induced by aminoglycosides. [18,19] The molecular mechanisms of ototoxicity is complex, many apoptotic pathways are involved in the HC death process as the increased level of reactive oxygen species (ROS). [20–23] When the amount of ROS exceeds the level that cells can repair, it becomes harmful. It has been reported that too much ROS can cause autophagy. [24,25] Cells have established protective mechanisms, such as autophagy, to remove ROS and maintain redox homeostasis. This protective mechanism is achieved by eliminating some of the dysfunctional mitochondria in cells and lowering ROS levels in cells. [26–28] A recent report showed that activating autophagy in HCs and HEI-OC1 cell lines not only reduces ROS levels, but also promotes cell survival. Conversely, down-regulation of autophagy in neomycin-damaged HCs and HEI-OC1 cell lines can significantly increase cell mortality, and rapamycin can reverse this process. [29] The mechanism of delayed ototoxicity induced by gentamicin has not been fully elucidated, but it may occur after the accumulation of toxic substances in cells reacts to gentamicin. The mechanism underlying this late-onset ototoxicity caused by gentamicin may be different from other ototoxic drug activation pathways such as cisplatin. This suggests that autophagy may play a dual role in the process of gentamicin exposure: promoting cell survival in the early stage and cell death in the late stage. Thorburn et al [30] reported that trehalose, a novel autophagy enhancer, can “delay” cell death after treatment with tumor necrosis factor-related apoptosis-inducing ligand by reducing the permeability of mitochondrial extracorporeal membrane. But this autophagy enhancer also causes mitochondrial apoptosis pathway. The authors suggested that autophagy could regulate the timing of mitochondrial outer membrane permeabilization in apoptosis. Therefore, autophagy may play essential roles in gentamicin-induced delayed toxicity.

In conclusion, aminoglycoside antibiotics cause autophagic cell death by interfering with the autophagic flow of auditory cells. In addition, increasing autophagic flow in vivo and in vitro by pharmacological means can increase the survival rate of auditory cells (Fig. 2). Deterioration of autophagy may be associated with delayed ototoxicity caused by aminoglycoside antibiotics.

Figure 2

Figure 2

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Autophagy and age-related hearing loss

Age-related hearing loss (ARHL), also known as presbycusis, is a complex disorder that results from the cumulative effects of aging on the auditory system. ARHL is defined as a progressive, bilateral, symmetrical age-related sensorineural hearing disorder that is pronounced at the high frequencies. As such, the health, societal and economic costs of ARHL are vast and increasing. However, compared with congenital and early-onset hearing loss, our understanding of the biochemical processes and molecular biology underlying this condition are limited. [31] The deterioration of auditory function over time is mainly related to the loss of stria vascularis, HCs and spiral ganglion cells in the cochlea of the inner ear. If the production of ROS in cells and the removal of ROS cannot reach a balance, it will lead to oxidative stress. Over time, it will lead to the occurrence of ARHL. [32] When ROS production exceeds the resistance of the antioxidant system, it causes irreversible damage to protein, DNA and lipids in auditory cells. In addition, antioxidant therapy has been shown to be effective in the treatment of ARHL and NIHL in mouse models. [33]

Autophagy has also been shown to play an essential role in development and disease. Evidence over the past century has shown that autophagy is directly involved in regulating the aging process. [34] Autophagy reporter analysis and gene expression studies of different species show that autophagy decreases with time. Genetic experiments that regulate the activity of autophagy genes in several short-lived models show that activating autophagy can be used as a strategy to promote longevity. In particular, studies conducted in model systems from yeast to mice have shown that conservative longevity model-induced longevity requires multiple ATG genes. Combining marker analysis of autophagy with gene expression analysis, it is generally indicated that the level of autophagy increases in longevity animals. These observations indicate that the increase of autophagy helps to prolong lifespan. Conversely, autophagy seems to be limited in the normal aging process, possibly in a tissue-specific manner, and involves selective autophagy types. A recent study has shown that injecting rapamycin, an autophagic promoter, into C57BL/6J mice can protect cells from oxidative stress and thus resist the development of ARHL, suggesting that rapamycin may be a potential drug for the treatment of ARHL (Fig. 3). [33] However, rapamycin can also cause many side effects, such as immunosuppression. Fortunately, many new rapamycin homologues, such as adenosine triphosphate competitive mammalian TOR inhibitors, have been developed. The side effects of these drugs in lymphocytes are significantly less than those of rapamycin, suggesting these new drugs are safer. Therefore, once these drugs are clinically proven to be effective and less toxic, they should be widely used.

Figure 3

Figure 3

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Autophagy and noise-induced hearing loss

According to the Centers for Disease Control, about a quarter of American adults are experiencing different forms of noise-induced hearing loss (NIHL). The human ear did not evolve in the presence of long and repetitive high-intensity noise stimuli from machines that surrounds people in work, leisure or combat settings in modern industrialized society, and thus is vulnerable to damage from these sounds. In the inner ear, auditory HCs convert sound signals into electrical signals through the swing of the sterecilia of the auditory HCs, and then electrical signals are transmitted through nerves to the auditory center of the brain, thus forming auditory sense. NIHL is usually considered to be a sensorineural hearing loss, because this dysfunction usually occurs in the inner ear. Occupational NIHL can have terrible consequences for individuals. Hearing loss will make it difficult for patients to communicate with others and increase their social pressure. Over time, it will lead to patients’ low mood, impaired self-esteem and even world-weariness. NIHL may be caused by short loud bursts or rising noise levels NIHL may be caused by short loud bursts or continuously rising noise levels. [35] This exposure can lead to HC damage in the cochlea, damage to surrounding supporting cells, and eventually lead to degeneration of related auditory nerve fibers. The degree of cochlear injury and hearing loss are closely related to the intensity and duration of noise. exposure. [36]

Like many disease processes, the pathogenesis of noise induced hearing loss has been proved to be closely related to environmental and genetic factors. It has been suggested that about half of the patients with NIHL are related to genetic factors. [37] Another key factor contributing to NIHL is oxidative damage to sensory HCs. Overproduction of ROS and reactive nitrogen is a common pathological factor in a variety of inner ear injuries in cochlear tissues and body fluids, including noise and ototoxic drugs. [38] Studies regarding lipid oxidation of 4-hydroxy nonylidene aldehyde and protein 3-nitrotyrosine (3-NT) showed that the oxidative stress of exogenous HCs was related to noise dose. Low levels oxidative stress induced by temporary threshold shift noise increased the light chain 3B (LC3B) of microtubule-associated protein in outer HCs, and blocked the oxidative damage induced by noise. In contrast, the overproduction of 3-NT and 4-hydroxynonenal by permanent threshold shift noise resulted in oxidative damage. Rapamycin prevents the death of HCs by blocking mammalian TOR complex 1, reducing the number of 3-NT, and increasing the level of LC3B. However, the reduction of LC3B by autophagy inhibitor 3-methylladenine or LC3B small interfering RNA can increase the level of 3-NT in outer HCs and promote the loss of HCs induced by noise (Fig. 4). [25] These results suggest that autophagy is an intrinsic cellular process that protects NIHL by reducing oxidative stress. In addition, these new observations suggest that activated autophagy is an untried but potentially valuable method for alleviating NIHL.

Figure 4

Figure 4

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Conclusions

Although we have made great progress in understanding of the molecular regulation of autophagy, the mechanisms underlying action of autophagy in the occurrence and development of auditory disease need further study. Targeted autophagy is undoubtedly a breakthrough in current clinical treatment, and some pre-clinical and clinical trials have shown that it is feasible to treat deafness-related diseases by regulating autophagy activity. To determine whether autophagy is an effective therapeutic target for auditory diseases; however, it will be necessary to develop better methods for autophagy detection as well as specific autophagy regulators that are more suitable for use in vivo. Such regulators would not affect other important cellular activities, but would play a specific role in the core action mechanism of autophagy. Other non-pharmacological approaches to inducing autophagy, such as diet restriction, weight-bearing exercise and gene therapy, as well as various hormones (eg, insulin) should also be explored further.

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Acknowledgments

None.

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Author contributions

XF designed and wrote the manuscript and participated in literature retrieval, manuscript drafting and writing of the main part in the manuscript. RC reviewed and modified the manuscript. All authors approved the final version of the paper.

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Financial support

None.

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Conflicts of interest

The authors declare that they have no conflicts of interest.

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Keywords:

autophagosome; autophagy; hearing loss; lysosome; microtubule-associated proteins light chain 3; mTOR; rapamycin; stress; treatment

Copyright © 2019 The Chinese Medical Association. Published by Wolters Kluwer Health, Inc.