Saving the hair cell: Investigating Apoptosis signal-regulating kinase 1 as a molecular target for preventing aminoglycoside induced hearing loss

Abstract

Aminoglycoside antibiotics are lifesaving medicines, crucial for the treatment of chronic or drug resistant infections. However, aminoglycosides can destroy the sensory hair cells of the ear. As a result, aminoglycoside treated individuals frequently experience a reduced quality of life, stemming from permanent high-frequency hearing loss and vestibular impairment. Currently, no alternate antibiotic substance has the same bactericidal profile or efficacy as aminoglycosides, and an adjuvant therapy capable of mitigating ototoxic outcomes does not exist.

In order to develop an otoprotective therapy, the mechanisms of aminoglycoside-induced hair cell death must be elucidated. With respect to this goal, the production of reactive oxygen species and subsequent c-Jun N-terminal kinase (JNK) and P38 mitogen-activated protein kinase (P38) phosphorylation has been extensively documented in aminoglycoside treated hair cells. However, strategies directly targeting ROS, JNK or P38 are limited by the importance of these molecules for normal cellular function. Notably, the upstream regulators of JNK or P38 have not been well studied in the ear. Therefore, this project aimed to elucidate the role of upstream regulator Apoptosis signal-regulating kinase 1 (ASK1) within the auditory system.

ASK1 is a key mediator of ROS induced JNK and P38 mediated disease. Importantly, ASK1 inhibition has previously been shown to reduce the pathological consequences of ROS, P38 and JNK signalling in varied disease models; without impeding the normal homeostatic cell function of these molecules. Therefore, ASK1 inhibition may represent a novel strategy for preventing aminoglycoside ototoxicity. However, the role of ASK1 as a mediator of drug-induced hair cell death has not been investigated. Moreover, the role of ASK1 in the development and function of auditory structures has not been explored.

This project first aimed to elucidate the importance of ASK1 in the inner ear by characterising the auditory phenotype of Ask1 knockout mice. Histology indicated normal development of cochlear structures and the auditory brainstem response (ABR) demonstrated that Ask1 knockout mice had hearing thresholds comparable to C57BL/6 controls. However, ABR peak analysis indicated that auditory signal transduction is faster in Ask1 knockout mice. In addition, the acoustic startle response showed Ask1 knockout mice to be hypersensitive to auditory stimuli. Combined, this data indicates that ASK1 is important for neuronal function and that Ask1 knockout mice have an auditory processing disorder.

This project then evaluated the importance of ASK1 as a mediator of drug-induced hair cell death. To test the hypothesis that ASK1 deficiency is protective against aminoglycoside-induced hair cell death, cochlear explants from Ask1 knockout and C57BL/6 mice were treated with neomycin in vitro.

After six hours of 1 mM neomycin treatment, immunohistochemistry demonstrated that p-JNK staining was reduced in Ask1 knockout explants when compared to C57BL/6 explants. Moreover, hair cell death was significantly attenuated in Ask1 knockout explants. This data provides robust evidence that ASK1 has an important role in the process of hair cell death, and that ASK1 inhibition could limit aminoglycoside-induced hair cell death.

Therefore, the final aim of this project was to test ASK1 inhibition as an otoprotective strategy. ASK1 inhibition significantly attenuated neomycin-induced outer hair cell death in C57BL/6 cochlear explants. Immunohistochemistry and western blot analysis suggested that ASK1 inhibition attenuated both P38 and JNK phosphorylation in neomycin-treated explants. Importantly, ASK1 inhibition did not impact the efficacy of the aminoglycoside antibiotics against P. aeruginosa in the broth dilution test. This indicates that ASK1 inhibition may mitigate ototoxic outcomes without impairing the primary aminoglycoside treatment.

Overall, this thesis represents the first aural characterisation of the Ask1 knockout mouse strain, providing valuable insight regarding the role of ASK1 in the auditory pathway. Moreover, this work has provided compelling evidence that ASK1 inhibition may be a useful strategy for the prevention of aminoglycoside induced hearing loss.