Endoplasmic Reticulum Stress in Hearing Loss

The endoplasmic reticulum (ER) plays important roles in coordinating protein biosynthesis and secretion in the cell. Accumulation of misfolded and/or unfolded proteins in the ER causes ER stress and the so-called unfolded protein response (UPR). The UPR alleviates ER stress through blocking protein synthesis and activating expression of chaperone genes, whereas prolonged UPR could induce cell death. Recent research has showed that ER stress and UPR are involved in hearing loss. Accordingly, animal experiments showed that chemical chaperones or ER stress inducers alleviate environment-related hearing loss, whereas ER stress inhibitor has been used to treat certain types of hereditary deafness. Further investigations are needed to fully understand the detailed mechanisms of how ER stress contributes to the loss of auditory function, which will help us to eventually develop ER-stress-related treatment of various types of deafness.


Introduction
The endoplasmic reticulum (ER) is a membranous tubular intracellular organelle that plays important roles in protein and lipid biosynthesis as well as calcium storage.Transmembrane and secretory proteins are synthesized and folded in the ER before being transported to their destinations.Unfolded or misfolded proteins are folded into correct conformations with the help of ER-residing chaperons such as Bip/GRP78, or subjected to degradation by the proteasome-dependent ER-associated protein degradation (ERAD) pathway [1].When there are too many unfolded or misfolded proteins accumulated in the ER, the ER becomes stressed and the so-called unfolded protein response (UPR) is activated [2][3][4].The UPR aims to alleviate ER stress through blocking protein synthesis and activating the expression of chaperone genes, whereas prolonged UPR could induce cell death [5,6].
At least three ER-stress-sensors are involved in UPR, the inositol-requiring enzyme 1α (IRE1α), the PKR-like ER kinase (PERK), and the activating transcription factor 6α (ATF6α) (Figure 1).All of the three sensors are transmembrane proteins normally associated with Bip/GRP78 at the ER.Unfolded or misfolded proteins that have accumulated in the ER sequester BiP from the ER stress sensors and activate UPR through different mechanisms: (1) After oligomerization and trans-autophosphorylation, IRE1α obtains endoribonuclease activity and induces the unconventional splicing of XBP1 mRNA into XBP1s [7][8][9].XBP1s encodes an active transcription factor whose target genes are involved in ER membrane biogenesis as well as ER protein folding [10,11].(2) PERK, which is structurally related to IRE1α, gains its kinase activity after oligomerization and trans-autophosphorylation. Activated PERK phosphorylates the α subunit of eukaryotic translational initiation factor 2α (eIF2α) at Ser51 [12,13].The phosphorylation of eIF2α attenuates the global protein synthesis, but enhances the translation of activating transcription factor 4 (ATF4) whose mRNA contains internal ribosomal entry sites (IRES) [14].ATF4 then induces expression of genes that are involved in ER function, reactive oxygen species (ROS) production, as well as cell death [15].Among the ATF4-target genes is C/EBP homologous protein (CHOP/GADD153), whose most well-known function is the induction of cell death [14,16].(3) ATF6α is a type-two transmembrane basic leucine zipper (bZIP) transcription factor.Upon release from BiP, ATF6α moves from the ER to the Golgi apparatus, where the cleavage by serine protease site-1 (S1P) and metalloprotease site-2 (S2P) releases the transcription-activating form of ATF6 [17][18][19].The activated ATF6 induces the expression of genes encoding several ER chaperons as well as XBP1 and CHOP [20].
ER stress is implicated in many diseases, including multiple types of neural diseases [36,37].More and more evidence suggests that ER stress plays an important role in hearing loss.In the following part, we will discuss the role of ER stress and UPR in different types of deafness.
ER stress is implicated in many diseases, including multiple types of neural diseases [36,37].More and more evidence suggests that ER stress plays an important role in hearing loss.In the following part, we will discuss the role of ER stress and UPR in different types of deafness.
Different ototoxic drugs activate different UPR pathways.For example, 3-NP treatment causes increased expression of ATF4 and CHOP, resulting in the apoptosis of lateral wall fibrocytes [39].In cultured cells derived from mouse organ of Corti (HEI-OC1), APAP or NAPQI treatment alters eIF2α phosphorylation as well as CHOP expression independent of ATF4 activation, and results in cell death [40].In contrast, aminoglycosides activate all three UPR pathways in cultured HEK293 cells [41].Interestingly, XBP1 could alleviate aminoglycoside-induced ER stress and hearing loss.Aminoglycoside-induced UPR was compromised and more severe hearing loss was observed in Xbp1 +/− mice compared with wild-type mice [41].Moreover, treatment of the chemical chaperone tauroursodeoxycholic acid (TUDCA) attenuated aminoglycoside-induced hearing loss in Xbp1 +/− mice, further supporting the hypothesis that UPR could protect drug-induced hearing loss [41].

ER Stress and Noise-Induced Hearing Loss
Prolonged exposure to high-intensity noise causes permanent hearing threshold shift called noise-induced hearing loss (NIHL).Research suggests that both HCs and SGNs are sensitive to noise damage [43].During NIHL, the protein level of Bip/GRP78, Caspase-12, and CHOP/GADD153 is elevated, suggesting a protective effect of UPR [44,45].The ER-residing chaperones Sigma-1 receptors (Sig-1Rs) are transcriptionally upregulated by the PERK/eIF2α/ATF4 pathway, and could ameliorate cell death signaling [46,47].Under ER Stress, Sig-1Rs stabilize IRE1α and activate the IRE1α/XBP1s pathway [48,49].Meanwhile, Sig-1Rs inhibit CHOP expression and decrease cellular toxicity [49].Sig-1R agonist cutamesine (SA4503) significantly reduces hearing threshold shifts and cell death in response to intense noise, whereas it has no effect on age-related hearing loss [50].At present, the detailed role of ER stress and UPR in NIHL awaits further investigation.

ER Stress and Age-Related Hearing Loss
Age-related hearing loss (ARHL), also referred to as presbycusis, is characterized by an elevation in hearing thresholds during aging.In the cochlea of aged mice, the expression of Bip/GRP78 is reduced, whereas the expression of CHOP is increased, suggesting that the UPR pathway is impaired and the cell death pathway is activated [51].Consistently, the cleavage of caspase-3, caspase-9, but not caspase-12, is increased in the aged cochlea [51].
Geranylgeranylacetone (GGA) is a widely-used HSP70 inducer; however, recently it was shown that GGA could also elicit UPR [52,53].Oral uptake of GGA attenuates ARHL in mice [54].Moreover, GGA has also been shown to ameliorate 3-NP-induced hearing loss and NIHL [55,56].The mechanism by which GGA protects auditory function has been attributed to its induction of HSP70, whereas the role of UPR in this process has not been examined and awaits further investigation.

ER Stress and Hereditary Hearing Loss
Gene mutations that affect ER stress and UPR have been shown to cause hereditary hearing loss (HHL).The most well-known deafness condition associated with impaired ER stress is caused by mutations in genes encoding WFS1 and connexins [57][58][59].Recently, it was shown that impaired ER stress is also involved in Usher (USH) syndrome [60,61].
Homozygous or compound heterozygous mutations in the human WFS1 gene cause Wolfram syndrome (WS), which is characterized by diabetes insipidus, diabetes mellitus, optic atrophy, deafness, and psychiatric illness [57,58].WFS1 gene mutations in WS patients cause a complete absence of WFS1 protein by nonsense-mediated mRNA decay (NMD), or instability and a strongly reduced half-life of WFS1 protein [68,69].In other words, WS-associated WFS1 mutations lead to loss of function of WFS1.Consistently, Wfs1 gene knockdown in cultured cells or Wfs1 gene disruption in mice caused elevated UPR and increased ER-stress-induced apoptosis [63,[70][71][72].It is worth noticing that Wfs1 disruption has different effects on different tissues in mice.In Wfs1 −/− mice, all three UPR pathways are activated in pancreatic cells, whereas only the IRE1α pathway is activated in the retina, and no ER stress is activated in other tissues examined such as the heart, skeletal muscle, and brown adipose tissue [71,72].WFS1 is widely expressed in multiple cell types in the inner ear, including HCs, SGCs, various supporting cells (SCs), and lateral wall fibrocytes [73].ER stress activation in the Wfs1 −/− inner ear has not been reported yet.
Heterozygous mutations in the WFS1 gene are identified in patients with WS [74] or nonsyndromic disease such as nonsyndromic dominant deafness DFNA6/14/38, which is one of the few low-frequency sensorineural hearing impairment (LFSNHI) [75,76].Research suggested that most heterozygous WFS1 mutations are likely small non-inactivating mutations [74,77].Interestingly, nine out of ten LFSNHI-associated WFS1 mutations affect the C-terminal protein domain of WFS1 [77].ER stress activation of LFSNHI-associated WFS1 mutations have not been reported at present.Nevertheless, WS-or diabetes-associated heterozygous WFS1 mutations have been shown to cause enhanced ER stress through ATF6α activation in transfected cells [78][79][80].
There is evidence that supports a possible link between ER stress and connexin expression and/or function.Treatment of cells with ER stress inducers resulted in decreased expression of connexins such as Cx36 and Cx43, as well as reduced gap junctional intercellular communication [93,94].Selective down-regulation of gap junction proteins might help to alleviate the burden of ER and prevent the transmission of 'stress' signals to adjacent cells, hence providing an important protective mechanism under ER stress [93].On the other hand, mutations in connexin genes have been shown to induce ER stress.For example, skin-disease-associated Cx31 mutants were shown to induce all three UPR pathways in transfected cells [95].ER stress is also activated in cataract-associated Cx50 mutant mouse lenses [96,97].Interestingly, different UPR pathways are activated by different Cx50 mutations.In Cx50G22R or Cx50S50P mutant mice, the IRE1α-XBP1 pathway is activated, while PERK-eIF2α-ATF4 and ATF6α pathways are largely unaffected [96].In contrast, in Cx50D47A mutant mice, the PERK-eIF2α-ATF4 pathway is activated, while the IRE1α-XBP1 and ATF6α pathways are not activated [97].
ER stress might also play a role in connexin-mutation-associated hearing loss.Several deafnessassociated Cx26, Cx29, Cx30, and Cx31 mutants have been shown to be trapped in the ER and are unable to transport to the plasma membrane in transfected cells [98][99][100][101][102].Moreover, Bip/GRP78 is upregulated in Cx31R180X-or Cx31E183K-overexpressed cells, suggesting that ER stress is elevated by these Cx31 mutations [101].It is worth noting that not all deafness-associated connexin mutants induce ER stress or are trapped in the ER.For example, deafness-associated Cx31(66delD) does not induce ER stress in transfected cells, suggesting that a mechanism other than ER stress is involved in these specific mutants [95].

USH Proteins and ER Stress
USH proteins are a group of proteins whose dysfunctions cause Usher syndrome, the most frequent form of inherited sensory deaf-blindness [103].USH is clinically classified into three types, namely USH1, USH2, and USH3, and more than ten USH proteins have been identified so far [104].Mutations in the USH genes are also associated with nonsyndromic hearing loss [104].USH proteins are expressed in HCs of the inner ear, especially on the stereocilia and at the ribbon synapses, where they bind to one another and form multiprotein complexes [104].At the cellular level, most USH gene mutations cause stereocilia and/or ribbon synapse deficits, and eventually result in hair cell death.However, the proximal cause of hair cell death remains elusive.
The first clue came from the finding that three CLRN1 (USH3A) pathogenic mutant proteins were mislocalized in the ER instead of on the plasma membrane in transfected cells [105].Similarly, USH proteins CDH23 (USH1D), harmonin (USH1C) and Myo7a (USH1B) were shown to preassemble into a complex at the ER in zebrafish hair cells, and defects in any one of the three USH proteins disrupt the complex and induce ER stress that triggers apoptosis [60].Thus ER stress might contribute to hair cell loss and provide a potential target for the treatment of Usher syndrome.
Recently, important progress was achieved using a mouse model erlong (erl) that harbors a point mutation (T208C) in Cdh23 gene.The erl mice suffered hearing loss through apoptosis beginning from postnatal day 27 (P27) [106].Further investigation showed that the PERK-eIF2α-ATF4-CHOP pathway is activated in erl mice, which eventually causes apoptosis of OHCs [61].Disruption of the Chop gene protects OHCs as well as auditory function in erl mice, further supporting the role of CHOP in ER stress-induced OHC apoptosis in erl mice [61].Most importantly, treatment of erl mice with ER stress inhibitor Salubrinal (Sal) delays the progression of hearing loss and preserves OHCs [61].Taken together, these data suggest that ER stress plays important roles in OHC loss and deafness in erl mice, and anti-ER stress drugs could serve an otoprotective purpose.

Conclusions and Perspectives
The role of ER stress and UPR in hearing loss is just starting to be appreciated.Several mechanisms have been proposed to contribute to DIHL, NIHL, and ARHL, including oxidative stress, calcium overload, inflammatory responses, and hereditary susceptibility [38,[107][108][109][110].Recent research has added ER stress and UPR to this list, although more details await further investigation (Table 1).Chemical chaperones or ER stress inducers have been shown to alleviate hearing loss, further confirming the protective role of ER stress in hearing.ER stress has also been shown to play a role in hereditary hearing loss (Table 2).WFS1 is a negative regulator in UPR, and WFS1 mutations result in elevated UPR and increase ER-stress-induced apoptosis, eventually causing hearing loss.Connexins and USH proteins are mostly transmembrane proteins that are important for hearing transduction.Dysfunctions of connexins or USH proteins might increase the burden of ER, hence resulting in ER stress that eventually leads to hearing loss.
Further investigation is needed to examine whether ER stress is also involved in hereditary hearing loss types other than the ones discussed above.More than 110 nonsyndromic deafness genes have been identified, which only represent a small portion of all deafness genes [111].At present, WFS1 is the only ER stress regulator whose mutations are associated with hearing loss.As novel deafness genes are identified, more ER-stress-related deafness genes will emerge.Additionally, similar to connexins and USH proteins, many deafness genes encode membrane proteins or secretory proteins, whose malfunction might increase the burden of ER and cause ER stress.
Present studies suggest that the detailed role of ER stress might differ in various types of deafness.ER stress seems to play a protective role in environment-related hearing loss (DIHL, NIHL, and ARHL), but a destructive role has been shown in certain types of hereditary hearing loss.If this is true, ER-stress-based treatment of different types of deafness should be different.In line with this, chemical chaperones or ER-stress-inducers have been shown to alleviate environment-related hearing loss, whereas ER-stress-inhibitors have been used to treat some types of hereditary deafness.Furthermore, different UPR pathways might be activated even in the same categories of deafness, suggesting that the role of ER stress in hearing loss is complicated, and that tailored ER-stress-based treatments must be developed.

Figure 1 .
Figure 1.Endoplasmic reticulum (ER) stress and unfolded protein response (UPR) in mammals.See main text for details.

Figure 1 .
Figure 1.Endoplasmic reticulum (ER) stress and unfolded protein response (UPR) in mammals.See main text for details.

Table 1 .
Summary of the roles of ER stress in environment-related hearing loss.

Table 2 .
Summary of the roles of ER stress in hereditary hearing loss.