Programmed cell death pathways in hearing loss: A review of apoptosis, autophagy and programmed necrosis

Abstract Programmed cell death (PCD)—apoptosis, autophagy and programmed necrosis—is any pathological form of cell death mediated by intracellular processes. Ototoxic drugs, ageing and noise exposure are some common pathogenic factors of sensorineural hearing loss (SNHL) that can induce the programmed death of auditory hair cells through different pathways, and eventually lead to the loss of hair cells. Furthermore, several mutations in apoptotic genes including DFNA5, DFNA51 and DFNB74 have been suggested to be responsible for the new functional classes of monogenic hearing loss (HL). Therefore, in this review, we elucidate the role of these three forms of PCD in different types of HL and discuss their guiding significance for HL treatment. We believe that further studies of PCD pathways are necessary to understand the pathogenesis of HL and guide scientists and clinicians to identify new drug targets for HL treatment.


| INTRODUC TI ON
Hearing loss (HL) is one of the most common sensory defects in humans. The hearing system is complex and depends on the comprehensive functions of many types of tissues and cells in the inner ear. Therefore, mutations in various genes have been proposed to be the cause of HL. It is estimated that 1-3 of every 1000 newborn children are deaf, and in nearly half of these cases, HL can be attributed to genetic factors. 1 The well-known types of acquired HL are ototoxic drug-induced hearing loss (ODIHL), age-related hearing loss (ARHL) and noise-induced hearing loss (NIHL). The pathological characteristics of each type of HL are not the same. The main mechanism of ODIHL is hair cell loss. 2 The loss of hair, spiral ganglion and vascular striated cells is involved in ARHL. 3 Noise-induced hearing loss is caused by excessive exposure to noise. 4 It involves two main mechanisms, namely mechanical damage and loss of hair cells and spiral ganglia. 4 Programmed cell death (PCD) appears to play a critical role in the development and diseases of the inner ear. When the nucleus of a cell is affected by severe damage, the initiation of PCD leads to irreversible changes, such as metabolic arrest, structural damage and function loss that can balance cell death and normal cell survival. Several forms of PCD have been found in eukaryotes, including apoptosis, autophagy, programmed necrosis, entosis, ferroptosis, lysosome-dependent cell death and parthanatos. [5][6][7][8][9] The contribution of apoptosis in the development of hearing loss has been long studied. Several studies have been conducted to decipher the molecular mechanisms underlying the roles of them in HL. Apoptosis is an ATP-dependent, enzyme-mediated, inherently programmed death of cells that are no longer needed or are a threat to the organism. 10 Apoptosis occurs when DNA molecule in a cell is beyond repair, when a cell receives stress signals from other cells, or when misfolded or unfolded proteins accumulate in a cell. The morphological manifestations of apoptosis include chromatin condensation, cell membrane blebbing, cell shrinkage and apoptotic body formation. 10 Autophagy is a conserved process of intracellular material turnover in eukaryotes. It is a key mechanism in the response of cells to extracellular or intracellular stress that aid in their survival under certain circumstances; for instance, autophagy protects cells against NIHL by attenuating oxidative stress. 11 However, overactivation of autophagy may result in cell death. 12 Specialized double-membrane vesicles, known as autophagosomes, encapsulate degenerating cytoplasmic organelles or cytosol and subsequently degrade them via the fusion with lysosomes. 13,14 Necroptosis can be initiated by several factors, and receptor-interacting proteins (RIPs) 1 and 3 are two key proteins involved in this process. Necroptosis can be morphologically characterized by increased cell volume, swollen organelles, ruptured plasma membrane and the subsequent intracellular content loss. 14,15 The contents of a ruptured necrotic cell in the interstitial space may trigger inflammation of the adjacent cells.
The pathology of HL has been studied extensively. 16,17 Recent findings suggest that cellular death mediated via PCD is an important mechanism in HL. Apoptosis and programmed necrosis always lead to cell death, whereas autophagy can lead to cell survival or death.
In normal cells, there is a delicate balance between apoptosis-inducing and apoptosis-inhibiting factors, and it ensures the survival and proliferation of cells. However, under stress, this balance can be disturbed. The activation of autophagy may play a protective role in the early stages of disease progression. Nevertheless, when the imbalance mediated by pathogenic factors becomes prominent, cells may activate the apoptotic or necrotic death process or over-activate autophagy leading to cell death. Thus, PCD is important in the development and maintenance of multicellular organisms, and the loss of PCD regulation can lead to diseases. Here, we focused on various causes of HL and the well-characterized cell death mechanisms (apoptosis, autophagy and necrosis) involved in HL.

| AP OP TOTI C PATHWAYS IN HL
Apoptosis is an active and highly ordered cell death process regulated by genes (including Bcl-2, p53 and c-Jun) and a series of enzymes (including caspases and endonuclease G (EndoG)) through intrinsic (mitochondrial), extrinsic (death receptor (DR)) and endoplasmic reticulum (ER) pathways. 18,19 Apoptosis plays an important role in maintaining the normal growth of an organism. The Bcl-2 family members are important for the regulation of apoptosis, including Bcl-2, Bcl-w, Bax, Bak, Bid and Bad. 20,21 The initiation of apoptosis depends on the activation of a series of caspases. Caspases can be divided into the following three categories: initiator (caspases initiator 2, 8, 9 and 10), executioner (caspases 3, 6 and 7) and inflammatory (caspases 1, 4 and 5). 22,23 When a cell is exposed to a fatal stress, apoptosis can be triggered by the initiator caspase 9 or 8 via the mitochondrial or DR pathways. Furthermore, the executioner caspases 3 and 7 are activated, causing the fragmentation of DNA, destruction of nuclear proteins, cytoskeleton and protein cross-linking, and expression of ligands in phagocytic cells. 24 In the caspase-independent pathway, apoptosis-inducing factor (AIF) and EndoG are released from the mitochondria, and they migrate to the nucleus to condense the chromatin (Figure 1). 22 ER stress is characterized by the accumulation of misfolded and unfolded proteins, and disruption of calcium and redox balances. 25 In multicellular eukaryotes, three upstream signalling proteins (IRE1, PERK and ATF6) act as pressure receptors, and they are activated by the level of unfolded proteins in the organelle cavities. 25 Cells can cope with ER stress by increasing the expression of chaperones and enhancing ER-associated degradation of misfolded proteins. 26 However, continued damage can lead to apoptosis (Figure 1). Studies have shown that oxidative stress can induce apoptosis via the DR and mitochondrial pathways. 27 Reactive oxygen species (ROS) are oxygen free radicals and non-radical substances, including hydroxyl radicals (OH-), su-

| Mutations of apoptosis-related genes leading to monogenic HL
The different chromosomal loci of nonsyndromic hereditary deafness are designated as deafness (DFN); letters A and B represent autosomal dominant inheritance (DFNA) and recessive inheritance (DFNB), respectively. Studies on mutant genes responsible for inherited progressive HL have suggested potential mechanisms underlying hair cell apoptosis. Table 1 lists three mutations in apoptotic genes that cause monogenic HL.

| DFNA5
DFNA5 is one of the mutated genes related to PCD that leads to sensorineural HL. So far, only intronic mutations have been reported to cause exon 8 skipping in patients with DFNA5-related HL. [28][29][30][31] The protein encoded by DFNA5 belongs to the gasdermin superfamily as it contains a gasdermin domain. A previous study reported that wildtype DFNA5 (wtDFNA5) had no effect on yeast cells, whereas mutant DFNA5 (mutDFNA5) led to cell cycle arrest. 32 In mammalian cells, the transfection of mutDFNA5 led mutDFNA5 to cell death, whereas the transfection of wtDFNA5wtDFNA5 could not. 33 Thus, HL caused by mutDFNA5 can be attributed to functional mutations. In a mutant DFNA5 cell line, the upregulation of different cytochrome c oxidase (COX) genes was found to be associated with cell death mechanisms under oxidative stress. 34 In the same research model, the downregulation of protein sorting-and folding-related mechanisms indicated that ER stress has a potential role in cell death induced by DFNA5 ( Figure 2A). 34

| DFNA51
DFNA51 is an inverted genomic duplication of 270-kb DNA, including the entire wild-type TJP2 that encodes the tight junction protein (ZO-2). ZO-2 belongs to the membrane-associated family of guanylate kinase homologs, and it contains 3 PDZ domains, 1 SH3 domain and 1 GUK domain. 35 ZO-2 binds to the C-terminal of the connective transmembrane protein and then connects to the actin in the cytoskeleton and regulates the location of different subtypes of cells by interacting with the signal transduction pathway molecules. 36 TJP2 is mainly expressed between the hair and supporting cells in the organ of Corti, and helps maintain the barrier between ductus perilymphaticus and ductus endolymphaticus. Its expression decreases with age. The pathogenic mutation gene is an inverted genomic duplication of TJP2 that results in the overexpression of TJP2, which leads to autosomal dominant nonsyndromic HL. The expression of TJP2 mRNA in patients with duplicate genes was approximately 1.7-fold higher than that in normal controls. The overexpression of TJP2 in vitro leads to a decrease in the phosphorylation and activation of glycogen synthase kinase 3β (GSK-3β). GSK-3β promotes cell death via the mitochondrial intrinsic apoptotic pathway, but it inhibits the DR-mediated extrinsic apoptotic pathway. 37 The results of real-time fluorescent quantitative polymerase chain reaction showed that even a slight increase in the expression of Bcl-w altered the expression of other Bcl-2 family members and the 18-kDa translocator protein (TSPO) may shift the overall steady-state balance towards apoptosis and thus result in HL. 38 However, the complete loss of TJP2 can lead to embryonic death; thus, TJP2 knockout was found to be lethal in mice ( Figure 2B). 39

| DFNB74
The mutations c.265 T > G and c.55 T > C in methionine sulfoxide reductase B3 (MSRB3) are related to autosomal recessive HL. The mutated gene is also known DFNB74. The gene has four isoforms.
Isotype A is located in the ER, and the other three isotypes (B, C and D) are located in the mitochondria. MSRB3 encodes a methionine sulfoxide reductase that is involved in the repair of oxidative damage proteins. In the organ of Corti in mice, the expression of MSRB3 is upregulated in the inner and outer hair cells, but it is downregulated in the supporting cells. MSRB3 mutations lead to the disruption of protein functions. This in turn leads to the accumulation of oxidative F I G U R E 1 Apoptotic signalling pathways. Factors such as ototoxic drugs, ageing and noise exposure, which lead to hearing loss, damage the antioxidant defence system of the cochlea and cause imbalance of oxidation-reduction in the inner ear. Reactive oxygen species (ROS) can directly induce the intrinsic apoptosis of cells. Moreover, they can induce the production of cell death ligands to mediate the extrinsic apoptosis process. ROS-induced intracellular protein damage can cause endoplasmic reticulum stress, which can lead to apoptosis (also known as BIM) molecules that promote apoptosis, and thus lead to HL ( Figure 2C). 40,41

| Aminoglycoside antibiotics
Aminoglycosides are broad-spectrum antibiotics, but their potential ototoxicity needs to be closely monitored. 42 The ototoxicity of aminoglycosides is irreversible because the hair cells of the cochlea cannot proliferate and recover. Aminoglycosides may damage the hair cells of the cochlea and type I sensory cells of the vestibule by triggering different apoptotic signals, thus resulting in HL and vertigo. 43 Mitochondrial pathways play a key role in aminoglycoside-induced apoptosis and may be the main target of these drugs. Aminoglycosides tend to accumulate in the mitochondria of hair cells. 44 Gentamicin directly inhibits protein synthesis in mitochondrial ribosomes and triggers the opening of mPTP that leads to the release of apoptotic factors. 44,45 In addition, L-carnitine promotes mitochondrial function, which can prevent the damage of the outer hair cells after the administration of gentamicin. 46 ROS has been identified as the main cause of HL mediated by aminoglycosides. 47 ROS induces mitochondrial damage, thereby F I G U R E 2 Schematics of three mutations that lead to monogenic hearing loss. A, DFNA5: the apoptosis-inducing region of DFNA5 is located in exons 2 and 6 of the N-terminal domain. Skipping exon 8 can change and shorten the C-terminal domain of DFNA5, reveal the apoptosis-inducing region and lead to apoptosis. B, DFNA51: overexpression of TJP2 induces apoptosis by activating glycogen synthase kinase 3β (GSK-3β). C, DFNB74: malfunctioning MSRB3 leads to the accumulation of oxidative damage proteins and reactive oxygen species (ROS), ultimately leading to endoplasmic reticulum stress and subsequent activation of endogenous apoptotic pathways leading to the activation of various pathways that lead to apoptosis. Aminoglycosides can accelerate the nonenzymatic formation of ROS by redox active iron complex, and induce the intracellular enzymatic reaction. 48 Additionally, a previous study demonstrated that dexamethasone, melatonin (MLT) and tacrolimus decrease the levels of ROS in GM-exposed explants. 49 Interestingly, the c-Jun-NH-terminal kinase (JNK) cascade reaction combines oxidative stress with apoptosis. 50 Through in vivo experiments, it has been shown that administration of an aminoglycoside leads to the activation of the JNK pathway, which triggers the apoptosis of cochlear cells. Accordingly, JNK cascade inhibitors, such as CEP-1347 and estradiol, can reduce the loss of hair cells after the administration of gentamicin. 51,52 However, Kalinec et al reported that gentamicin ototoxicity is mediated by the inhibition of the JNK pathway. 46 Evidently, there is no consensus on whether the signalling enzyme is activated by gentamicin ototoxicity.
Hair cells are not the only drug targets. Aminoglycosides also have effects on stria vascularis, including thinning of the tissue and reduction of marginal cells. 53,54 The degeneration of spiral ganglion cells after aminoglycoside treatment may be attributed to the loss of hair cells innervated by the ganglion cells. 55 However, some studies have shown that the spiral ganglion can be affected without obvious damage to the hair cells. 56,57 This suggests the complexity of the damage pattern of aminoglycoside antibiotics.

| Cisplatin
The ototoxicity of cisplatin is well known. 58 The ototoxic effects of cisplatin can be divided into two categories. The first is the reversible inhibition of conduction current, voltage-dependent calcium current in hair cells and the current response in stria vascularis. 59 Another persistent toxic reaction induces irreversible changes in cochlear morphology, thus resulting in irreversible, bilateral, high-frequency HL. 60 Compared with the inner hair cells, the outer hair cells, 61  Cisplatin increased the production of ROS in the inner ear. 68 It seems that one of the important sources of these ROS is nicotinamide adenine dinucleotide phosphate (NADPH) oxidase 3, which is a type of superoxide that produces NADPH oxidase. It is highly expressed in the Corti organ, 69 and its level increases after cisplatin treatment. 69,70 Other NADPH oxidases are also important in the production of ROS in response to cisplatin ototoxicity. 71 Excessive ROS production may damage the antioxidant defence capacity of cochlear cells. p53 is activated in response to oxidative stress to regulate the expression of genes (eg Bax) that control DNA repair and cell death. 72 Bax can interact with voltage-dependent ion channels in mitochondria, which mediate the release of cytochrome c and have the effect of apoptosis. 70

| Age-related Hearing Loss (ARHL)
The prevalence of ARHL is expected to rise with the increase in the ageing population. 85 93 It has been demonstrated that in ARHL models of mice, rats and gerbils, apoptosis occurs through the caspase-dependent pathway, and involves the Bcl-2 family proteins. [94][95][96] Immunohistochemical analysis of the cochlea of ageing CBA/J mice showed an increase in the phosphorylation (ie activation) of JNK and p38 MAPK in outer hair cells. 94 In addition, the same study also proved the release of cytochrome c, activation of caspase 9 and translocation of Endo G in the hair cells of ageing mouse.
ROS play an important role in ARHL. 97  In addition, C57BL/6J mice provided an antioxidant diet (α-lipoic acid, coenzyme Q10, NAC) had a much lower ABR hearing threshold than that of control mice. 93 Nevertheless, Sha et al kept CBA/J mice on a long-term diet from 10 to 22 months of age and claimed that foods rich in vitamins A, C, E and alpha lipoic acid did not delay or reduce ARHL. 94 Accordingly, Keithley et al found that transgenic mice expressing SOD2 yielded outcomes that were contrary to expectations, while the HL in mice who were 20 months old was more prominent than that in the parent strain of B6 mice. 101 These results suggest that mitochondrial ROS may be a factor in ARHL, but there are other factors involved as well.
Mitochondria are particularly susceptible to the accumulation of genetic or environmental damages because-unlike the nucleus-mtDNA is regularly replicating independent of cell cycle replication and lacks an effective DNA repair system and protective histones.
Therefore, the total number of its DNA mutations is higher than that of the accumulation of mitochondrial DNA mutations are thought to cause age-related degenerative diseases, 102 and an increase in mitochondrial DNA mutations in human cochlear tissue has also been observed. 103 The same mechanism was proposed in the mouse ARHL model. 104 However, the exact pathway of apoptotic activation caused by ARHL has not been clearly defined. In fact, it is possible to activate multiple pathways at the same time because ARHL is the product of a multifactorial process.

| Noise-induced HL (NIHL)
Noise is also the main cause of HL. 106 After noise exposure, there are two main mechanisms that cause cochlear damage. The first is direct mechanical damage that leads to the loss of hair cells through the mechanical destruction of cilia, and to the damage of supporting and sensory cells. 107 Another mechanism involves the biochemical pathway that leads to cell death through apoptosis or necrosis. It has been shown that apoptosis is the key mediator of noise-induced HL. After noise exposure, an increase in chromatin condensation and levels of apoptosis markers [108][109][110][111][112] such as caspase 3, 8 or 9, 113 tumour necrosis factor receptor, 111 and Bcl-2-associated death promoters (Bad) 114  However, these factors individually have limited protective efficacy.
These observations are consistent with the suggestion that noise may induce cell death via both caspase-dependent and caspase-independent apoptosis. 117 After noise exposure ends, ROS or other similar reactive species levels are generally increased. 120 ROS were observed to be present in the cochlea for an extended period of time following noise exposure. 121 These species were responsible for the morphological observations of delayed and sustained damages. 121 Consistent with the hypothesis of ROS formation, antioxidant molecules can be used for protection, such as water-soluble coenzyme Q10, 122 NAC, 119,123 D-methionine 124 and GSH, 125 which will decrease the amount of apoptosis in hair cells after noise exposure. ROS formation can activate the JNK signalling pathway. 126 In the noise-damaged guinea pig model, JNK mediates apoptosis, 110 and blocking of the JNK pathway has a protective effect on noise. Mice exposed to noise contained fewer apoptotic cells than those in the control group, when they were fed with JNK inhibitors such as all transretinoic acid and CEP-1347 (small molecules derived from indole-carbazole K252a). [127][128][129] Another study reported that blocking the JNK pathway with locally delivered D-JNK-1 through the round window membrane can prevent hair cell death and permanent NIHL. 110 In addition, the increase of free Ca 2+ in the outer hair cells, or the activation of Ca 2+ and calmodulin-controlled calcineurin may trigger the apoptosis or necrosis pathway without ROS. 130,131 Calcium metabolic disorders and free radicals can also cause ER stress. Severe ER stress is more likely to induce the expression of CHOP 132 and would lead to ER stress-related apoptosis based on the downregulation of the expression of antiapoptotic proteins such as Bcl-xL. 133 Glucocorticoid-induced leucine zipper protects the cochlea from ER stress-induced apoptosis following noise exposure by reducing chop and regulating ER stress-related apoptosis proteins. 134

| AUTOPHAG I C PATHWAYS IN HL
Autophagy is a protective mechanism triggered to limit pathological changes. In all eukaryotic cells, autophagy processes unnecessary or dysfunctional cell components such as damaged organelles and misfolded proteins 135 through highly regulated processes. 136,137 Autophagy is a process that mediates the formation of a bimembrane autophagic body that surrounds the damaged organelle or other cytoplasmic components. 138 The core molecule of autophagy regulation is the rapamycin kinase mammalian target Some studies showed that autophagy plays a role in the prevention of hearing impairment, such as NIHL and ODIHL. 11,143 Previous studies have shown that ROS has the ability to induce autophagy in auditory cells. 11 is the first microRNA mutation that has been reported to be related to human deafness. 149 The decreased expression of mi-croRNA-96 may directly upregulate the expression of ATG7.
Excessive activation of autophagy induces degeneration and death of neurons. 150

| THE PROG R AMMED NECROS IS PATHWAYS IN HL
Programmed necrosis is a type of regulatory cell death caused by microenvironmental disorders inside and outside the cells and is de- and necrosis of cells. 158 These findings suggest that a balance between apoptosis and necrosis is required for noise-induced death of outer hair cells, which is regulated by caspase 8 and RIP kinase.
Choi et al found that treatment with Nec-1, a selective RIP1 inhibitor, significantly inhibited cisplatin-induced cell death in HEI-OC1 cells, while the use of ZVAD did not change cisplatin-induced cell death in HEI-OC1 cells. 159 Their results suggested that RIP3-dependent cell necrosis may mediate cisplatin ototoxicity. 159 Douglas et al studied the ototoxicity of aminoglycosides and cisplatin in a murine model 160 and suggested that the main form of hair cell death induced by aminoglycosides and cisplatin in vitro was mediated by caspase-dependent apoptosis, without any effects from necrosis. In vivo, Nec-1 was used to inhibit RIP1-mediated necrotic disease and reduce HL induced by kanamycin and cisplatin. 160 These results suggest that F I G U R E 4 Signalling pathway of programmed necrosis in hearing loss. When a ligand binds to tumour necrosis factor receptor (TNFR), a combination of TNFR-associated death domain (TRADD) and receptor-interacting protein (RIP) 1 increases the level of RIP3 and induces self-and transphosphorylation, which is followed by the oligomerization of phosphorylated RIP3. Active RIP3 catalyses the phosphorylation of the mixed lineage kinase domain-like protein (MLKL), thus resulting in the formation of MLKL oligomers and in translocation to the plasma membrane. Through the reversal mechanism, specific phosphatidylinositol phosphates are combined that lead to plasma membrane permeability and eventually cell necrosis the harmful factors (ototoxic drugs, ageing and noise exposure) can induce cell death via different PCD pathways. However, the crosstalk between these types of death pathways need to be investigated in future studies.

| CON CLUS IONS
The auditory system is

CO N FLI C T O F I NTE R E S T
The authors declare no conflict of interest.

DATA AVA I L A B I L I T Y S TAT E M E N T
Data sharing is not applicable to this article as no new data were created or analysed in this study.