Aminoglycoside-Induced Hair Cell Death and Potential Therapies

Introduction

Hearing loss is the third most common disorder in the United States, being more prevalent than diabetes or cancer (Blackwell et al., 2014). Our ability to hear is dependent on hair cells that can translate the sounds we hear into electrical impulses that can be interpreted by the brain. When sound waves enter the cochlea located in the inner ear, they deflect the apical portion of hair cells, also known as stereocilia. Stereocilia are connected to one another via tip links.

Excitatory deflection results in the "tilting" of the stereocilia in a domino effect, which "pulls open" the mechanotransduction (MET) channels located at the tips of the stereocilia. Potassium ions flux inside hair cells through the open MET channel, resulting in inward depolarizing currents. This can stimulate the release of glutamate onto innervating afferent neurons, resulting in postsynaptic excitatory action potentials (Nordang et al., 2000; Oestreicher et al., 2002).

Hearing loss can occur due to multiple factors including noise, genetics, age, and chemicals such as aminoglycosides. Aminoglycoside antibiotics are highly effective at treating tuberculosis and Gram-negative bacterial infections associated with cystic fibrosis and sepsis (Forge and Schacht, 2000). However, aminoglycosides have the unfavorable effect of ototoxicity and nephrotoxicity; they cause damage to hair cells in the inner ear and to proximal tubule cells in the kidney, respectively. Cochlear hair cells are responsible for hearing, and vestibular hair cells are responsible for balance. Twenty percent of patients taking lifesaving aminoglycosides suffer permanent hearing loss and/or balance disorders (Ariano et al., 2008; Al-Malky et al., 2015; Garinis et al., 2017). Hearing loss results in socioeconomic burdens of nearly 1.4 million dollars throughout the lifetime of a child born deaf, and greater than $350,000 for an afflicted adult (Mohr et al., 2000). Despite this, aminoglycosides are commonly used worldwide, with nine aminoglycosides approved by the US Food and Drug Administration. The popular use of aminoglycosides, despite side effects, is due to their quick bactericidal activity, chemical stability, low costs, and low occurrences of bacterial resistance (Puopolo and Eichenwald, 2010; Tsai et al., 2014).

Nearly all cells take up aminoglycosides, but most cells quickly sequester and remove the aminoglycosides from their cytoplasm (Dai et al., 2006). This is not the case for inner ear hair cells, nor renal proximal tubule cells, which both retain aminoglycosides for increased amounts of time (Dai et al., 2006). Hair cells and proximal tubule cells are preferentially loaded with aminoglycosides (Humes, 1999). The retention of aminoglycosides and the cells' higher metabolic rates are likely the reasons for the cells' increased susceptibility to aminoglycoside-induced damage (Dai et al., 2006). This review will focus on the entry of aminoglycosides into hair cells, and the potential therapies that include the attenuation of aminoglycoside-induced hair cell death.

Entry of Aminoglycosides into Hair Cells

Aminoglycosides are rapidly taken up by hair cells, primarily via the MET channel (Marcotti et al., 2005). The liquid surrounding hair cells, called endolymph, has a strong positive membrane potential, whereas the cytoplasm of hair cells has a strong negative membrane potential (Pickles, 2012). This creates a strong electrochemical force that drives cations, including aminoglycosides, through open non-selective cation channels, such as the MET channel (Pickles, 2012). The MET channel is a complex composed of TMC proteins forming the pore (Pan et al., 2018), myosin VIIA (Kros et al., 2002), and cadherin-23/protocadherin-23 proteins forming the tip links (Vu et al., 2013), amongst other proteins. When any of these are mutated, MET channel conductance is decreased, which reduces aminoglycoside uptake. Fluorescently-conjugated gentamicin (Gentamicin-conjugated Texas Red, or GTTR) has been localized at the apical membranes of hair cells in the hair bundles before being taken up into hair cells (Steyger et al., 2003). Aminoglycosides also enter through other ion channels, such as various TRP channels. One of the reasons hair cells and proximal tubule cells are particularly susceptible to aminoglycoside-induced death is due to their uniquely high expression of TRP channels (Steyger and Karasawa, 2008).

Aminoglycosides are also endocytosed at the apical membranes of hair cells from the endolymph into the cytoplasm. From there, aminoglycosides are rapidly sequestered within lysosomes (Hashino et al., 1997; Hailey et al., 2017). Interrupting the endocytosis pathway (effective in the kidney) surprisingly increases aminoglycoside-induced hair cell damage. This is because endocytic and non-endocytic pathways compete, so aminoglycoside loading will not be reduced, and endocytic pathways actually result in rapid sequestering into lysosomes, mitigating damage. Blocking the MET channel prevents aminoglycoside entry, providing protection, but simply reducing aminoglycoside entry results in protection via sequestering into lysosomes (Hailey et al., 2017).

Mechanisms of Aminoglycoside-Induced Ototoxicity

Aminoglycosides are effective bactericidal agents that interrupt genetic translation by inducing ribosomal misreading in bacteria (Cox et al., 1964; Davies and Davis, 1968). Aminoglycosides also bind to cytosolic rRNA in the inner ear and kidney of eukaryotes, which inhibits protein synthesis in those locations as well. This leads to ribotoxic stress-induced apoptosis (Francis et al., 2013).

Aminoglycosides also bind to cytosolic proteins and inhibit their normal function (Karasawa et al., 2010). This includes calreticulin and CLIMP-63. Calreticulin is a chaperone protein of the endoplasmic reticulum (Horibe et al., 2004; Karasawa et al., 2011) responsible for protein folding and degradation (Williams, 2006). Calreticulin is highly expressed in hair cell stereocilia (Karasawa et al., 2011). Aminoglycosides act as competitive antagonists, preventing calcium from binding to calreticulin, resulting in a loss of calreticulin function (Karasawa et al., 2011). This either disrupts the chaperoning, calcium buffering, or regulation of calreticulin activity, and these disruptions may result in cytotoxicity (Bastianutto et al. 1995; Mesaeli et al., 1999). CLIMP-63 is another endoplasmic reticulum protein (Karasawa et al., 2010) responsible for anchoring microtubules to the endoplasmic reticulum (Sandoz and van der Goot, 2015), found in the murine organ of Corti. The organ of Corti is the functional unit in the inner ear where hair cells are located. Aminoglycosides bind to CLIMP-63, which then binds to 14-3-3 proteins (Karasawa et al. 2010). 14-3-3 proteins bind to and inactivate MDMX, a negative regulator of p53. p53 is a well-known tumor suppressor protein (Okamoto et al., 2005). In total, aminoglycoside binding to CLIMP-63 leads to subsequent apoptosis dependent on p53 activation.

Aminoglycosides may also enact the transfer of calcium from the endoplasmic reticulum to the mitochondria via IP3 receptors (Esterberg et al., 2013). This increases mitochondrial calcium concentrations, which results in elevated levels of oxidation and subsequent accumulation of cytotoxic levels of reactive oxygen species (Esterberg et al., 2016). Other mechanisms of aminoglycoside-induced hair cell death include the activation of the stress-induced c-Jun N-terminal kinase pathway (Ylikoski et al., 2002), the release of cytochrome C from the mitochondria into the cytosol, which activates caspases (Matsui et al., 2004), and excess glutamate signaling leading to an overabundance of calcium influx through NMDA channels (Sheets, 2017).

Attenuation of Aminoglycoside-Induced Hair Cell Death

Aminoglycoside-induced hair cell death can be attenuated via prevention of uptake or interruption of intracellular mechanisms leading to apoptosis. Prevention of uptake has been studied thoroughly. A screen of 1,040 FDA-approved drugs identified eight compounds that protect against exposure to both neomycin and gentamicin. All eight drugs share a similar quinoline structure and are thought to reduce aminoglycoside uptake into hair cells (Ou et al., 2012). In another study, four bisbenzoquinoline compounds (plant alkaloids) were identified from a screen of 502 natural compounds to be otoprotective against acute neomycin and continuous gentamicin exposures. These included berbamine, E6 berbamine, hernandezine, and isotetrandrine – with E6 berbamine being the most protective. All four compounds reduced GTTR and FM 1-43x (dye that indicates MET channel functionality) uptake, suggesting a blocking of aminoglycoside uptake through the MET channel (Kruger et al., 2016). In a subsequent study, the two otoprotectant compounds d-Tubocurarine (dTC) and berbamine were tested in zebrafish and neonatal mice. Berbamine and dTC, both MET channel blockers, protect zebrafish hair cells from neomycin- and gentamicin-induced hair cell death and protect mouse cochlear hair cells from gentamicin-induced hair cell death. Berbamine is toxic to the mouse cochlear hair cells at high concentrations, while dTC is not (Kirkwood et al., 2017). In another study looking at additional mechanisms, two lead otoprotectants identified are thought to protect by blocking calcium entry through NMDA channels. Three other lead otoprotectants work in various ways, with one blocking the MET channel but not GTTR uptake, one reducing GTTR uptake but not blocking the MET channel, and one that doesn't do either. These results indicate protection downstream of aminoglycoside entry (Kenyon et al., 2017).

If the uptake of aminoglycosides is not prevented, attenuation of aminoglycoside-induced hair cell death can still occur through the interruption of intracellular apoptotic mechanisms. Aminoglycosides, such as apramycin, have been developed with minimal affinity for eukaryotic mitochondrial ribosomes, while maintaining their bactericidal activity (Matt et al., 2012). Another study modified the amine groups on sisomicin, a precursor of gentamicin, and developed a drug called N1MS that is significantly less ototoxic, but remains bactericidal (Huth et al., 2015). Antioxidants such as N-acetylcysteine, D-methionine, and edaravone have been used to reduce the buildup of cytotoxic levels of reactive oxygen species in mammals post-aminoglycoside exposure (Somdas et al., 2015; Campbell et al., 2016; Turan et al., 2017). CEP-1347, an inhibitor of JNK signaling, has been used to attenuate neomycin- and gentamicin-induced hair cell death (Ylikoski et al., 2002). Protection can occur downstream of aminoglycoside entry via multiple different interventions.

Conclusion

Aminoglycosides are highly effective bactericidal agents, but they have pronounced ototoxic and nephrotoxic effects. Despite less toxic alternative antibiotics, aminoglycosides continue to be used due to their low cost, chemical stability, and low occurrence of bacterial resistance. Therefore, attenuating aminoglycoside-induced hair cell death is of great importance. Work has been done to identify methods of aminoglycoside entry into hair cells, mechanisms of aminoglycoside ototoxicity, and attenuation of aminoglycoside-induced hair cell death. Promising results have led to the identification of many otoprotective agents that may decrease the risk of permanent hearing loss for patients taking lifesaving aminoglycosides.

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