The majority of the 154 autophagy- inducing and/or -inhibiting compounds had no significant effect on HCs after GM treatment. An example is illustrated in Figure 4. When the highest concentration (10 μM) of autophagy compound was added to the micro-explant HC numbers were similar to that seen for negative control micro-explants cultures in media alone. Adding three different concentrations of autophagy compound plus 200 μM GM produced HC survival rates comparable to those observed with GM alone.

Eighteen autophagy compounds showed statistically significant protection of HCs in micro-explants treated with both the compound and gentamicin (Table 1). Within the group of protective compounds the observed protective effect could be divided into strong, medium and slight protection (Table 2). Strong protective compounds exhibited significant HC survival on all 3 days, varying in concentration (Figure 5A). Medium protective compounds showed improved HC survival on 2 days with one or two concentrations (Figure 5B), while slightly/mildly protective compounds reduced HC loss on D1 and only for 10 μM concentration (Figure 5C). There were two test substances that exhibited a bimodal trait i.e., significant protection at low concentrations, but enhancing GM damage at the highest concentration. Bortezomib and Brefeldin A exhibited significant protective traits at 0.1 μM, Brefeldin A also with 1 μM on Day 2, they both demonstrated significantly enhanced early HC loss due to gentamicin at 10 μM (Figure 6). We observed this “push-pull” effect with tyrosine kinase inhibitors in previous studies (Ryals et al., 2017).

One autophagy compound BGT 226 showed HC decrease when treated with 10 μM of the compound alone (even in the absence of gentamicin). This was significant at 48 and 72 h (Figure 7).

Proteasomes are protein complexes that degrade unneeded, misfolded or damaged proteins by proteolysis. Misfolded proteins trafficked from the Golgi to the ER are retained in the ER and retro-translocated to the cytosol in vesicles (Omari et al., 2018). Along with damaged proteins, they are tagged for proteasome proteolysis by ubiquitins (Taxis et al., 2002). Autophagy is thought to co-operate with the ubiquitin-proteasome system in responding to cell injury (Ji and Kwon, 2017). The proteasome is known to protect cells from degeneration by removing damaged proteins (Jones and Tepe, 2019), but an overactive proteasome can enhance tissue damage (Meller, 2009; Chen et al., 2010). Six compounds out of the 18 protective compounds were proteasome inhibitors.

The library had twelve inhibitory compounds targeting the proteasome pathway and of which six were identified protective in our screen. These results strongly suggest that the proteasome overactivity contributes to HC damage. Cheng et al. (2016) identified that Atoh1 (Atonal BHLH Transcription Factor 1) was regulated by the ubiquitin-proteasome system and hence ubiquitin proteasome pathway appears necessary for hair cell fate determination and survival. Also, we recently reported that multiple proteins of the ubiquitin-proteasome system are up-regulated in the cochlea following noise damage (Jongkamonwiwat et al., 2020), suggesting the possibility of a similar involvement in noise-induced HC damage associated with protein abundance and protein misfolding.

In autophagy, mature autophagosomes are transported to lysosomes along microtubules with force generated by the actin cytoskeleton. Microtubules are also involved in the transport of other cellular constituents, cell structure and cell migration. The library had twelve compounds associated with autophagy, microtubules and actin of which four inhibitors of were protective.

Calcium regulates autophagy at several different points in the process (Bootman et al., 2018). Increases in intracellular calcium, from calcium channels, calcium uptake or release from mitochondria, can induce autophagy. Conversely, calcium release leading to entry into mitochondria increases ATP production, which inhibits mitochondria (Kondratskyi et al., 2013). However, calcium also plays multiple additional roles in cells. Loss of calcium homeostasis is a major factor in cell injury and death through multiple mechanisms include apoptosis and necrosis (Dong et al., 2006). Intracellular calcium is tightly regulated in part to prevent the accumulation of excessive levels. HCs in particular contain high levels of calcium buffering proteins and express multiple calcium channels and transporters. Calcium subserves multiple normal functions of HCs (Heller et al., 2002). A number of studies have highlighted the role of calcium in HC damage, including release of calcium from the endoplasmic reticulum and mitochondria. It has been suggested that differences in HC vulnerability to damage along the length of the cochlea are related to variation in the ability to handle calcium load (Fettiplace and Nam, 2019). It has been suggested that calcium channel blockers should be investigated as therapeutic agents for acquired hearing loss (Naples, 2017). In this screen, the library had fourteen compounds that influence intracellular calcium levels and four were found to be protective.

The expected results of these inhibitors would be to reduce intracellular calcium. While this strongly supports a critical role for calcium in HC damage, it cannot be attributed with any certainty to the regulation of autophagy.

The PI3K/AKT/mTOR is an evolutionally conserved cascade that integrates signals from multiple pathways, including nutrients (e.g., amino acids and glucose), growth factors (e.g., EGF, PDGF, IGF-1), hormones (e.g., leptin), and stresses (e.g., starvation, hypoxia, and DNA damage) to regulate a wide variety of eukaryotic cellular functions, such as autophagy, translation, transcription, protein turnover, metabolism, energy balance, and stress response, as well as cell proliferation, growth, differentiation and survival. mTOR can also be activated independently of PI3K and AKT. The library contains 25 compounds related to PI3K/mTOR and three inhibitors were protective.

Because of the diverse rolls of the PI3K/AKT/mTOR pathway, the mechanism by which these inhibitors protect HCs is not clear. mTOR reduces autophagy by negatively regulating several key molecules (Kim and Guan, 2015), so the promotion of autophagy is one possibility. However, in our prior screen of kinase inhibitors (Ryals et al., 2017), one EGFR inhibitor, two PDGF receptor and two AKT inhibitors were found to be protective. This result is consistent with a damaging role for the PI3K/AKT/mTOR pathway, which is activated by growth factors. However, one of the autophagy compounds, BGT 226, showed a “push pull” relationship with a toxic effect in the highest concentration leading to HC death. Deletion of either AKT1 or AKT2 has been shown to enhance HC death due to aminoglycosides (Brand et al., 2015). It is possible that the three PI3K/mTOR inhibitors preferentially target PI3K/mTOR mediation of stress responses. Alternatively, they may target one of the several mTOR responses that are independent of AKT.

Aurora kinase A is critical to the process of mitotic spindle formation and separation, and thus of cell division. Inhibition of Aurora kinase A blocks cell proliferation and can induce autophagy (Zou et al., 2012). In our screening assay two inhibitors of Aurora kinase inhibitors showed a protective effect:

Overall the library had 20 compounds related to the Aurora kinase pathway and only two compounds were protective. This is consistent with the finding that maintaining the fully differentiated state is critical for HC survival and that forced cell cycle re-entry in differentiated, post-mitotic cells in response to stress can lead to mitotic catastrophe and cell death (Weber et al., 2008; Sharma et al., 2017). This provides strong evidence that one aspect of gentamicin-induced HC damage may be induction of the cell cycle.

NFkB Inhibitor: BAY 11-7082. The role of immune responses and inflammation in HC damage is increasingly recognized (Frye et al., 2019). Damage to HCs results in the release of inflammatory mediators such as the inflammatory cytokine TNFα (Tan et al., 2016). Damaged cells can also release cellular constituents that serve as damage-associated molecular patterns (DAMPs). Damps in turn can activate innate immune receptors such as the Toll-like receptors (TLRs), leading to the production of inflammatory cytokines such as IL1β and TNFα, as well chemokines that attract immune cells including macrophages. Many inflammatory responses are mediated by the transcription factor NFkB, which can also stimulate autophagy. An inhibitor of this factor was protective in the screen. This result supports a role for inflammation in ototoxic HC damage.

An inhibitor of protein trafficking was protective, Brefeldin A. Brefeldin A is an inhibitor of protein trafficking between the endoplasmic reticulum and the Golgi apparatus (Chardin and McCormick, 1999). These results suggest that the movement of intracellular elements, possibly including autophagosomes, contribute to ototoxic HC damage.

The majority of the compounds that proved to be protective to HCs against gentamicin damage would be expected to reduce autophagy. While several of these compounds have complex effects, the fact that multiple autophagy inhibitors reduced HC loss argues for an autophagy role in promoting HC damage due to GM. Levano and Bodmer found that mutation of the Stat1 gene protected HCs from ototoxic damage in part by modulating autophagy genes so as to reduce autophagy (Levano and Bodmer, 2015).

Autophagy is often considered to be protective of cells under stress, but it has been shown that excessive activation of autophagy can lead to autophagic cell death, or autosis (Liu and Levine, 2015). The high dosage of GM used in this study may have induced excessive autophagy. Apoptosis, necrosis and necroptosis have been shown to participate in HC death (Ruhl et al., 2019). However, autosis has not previously been linked to HC damage. Autosis is characterized by nuclear shrinkage with focal concavities and ballooning of the perinuclear space. These characteristics are visible in some published electron micrographs of aminoglycoside-damaged HCs (Lang and Liu, 1997).

While most autophagy inhibiting compounds were protective, we also observed protection with compounds that would be expected to enhance autophagy. Again, while these compounds have complex effects, the results support the concept that autophagy can be protective against aminoglycoside damage. It seems likely that autophagy plays a complex role in ototoxic HC damage, such that manipulation of different aspects of the process can have differing effects. The interactions of identified compounds with the autophagy machinery warrants further mechanistic studies because these candidate leads may employ one or more of several mechanisms. Therefore, differences in their effectiveness may vary with differences in the cellular processes involved and aminoglycoside impact. Given the complexity, it would not be expected that all compounds in a category would have equivalent effects on cell damage and survival.