O'Sullivan, ME; (2015) Investigation of mitochondrial m.1555A>G aminoglycoside hearing loss. Doctoral thesis , UCL (University College London).

Abstract

The mitochondrial DNA mutation, m.1555A>G, predisposes to severe hearing loss following aminoglycoside antibiotic exposure. The m.1555A>G mutation can also lead to hearing loss in the absence of antibiotic exposure termed non-syndromic m.1555A>G hearing loss. Two distinct concepts of mitochondrial dysfunction related to the m.1555A>G mutation have been postulated - mRNA misreading and 12S m⁶₂A rRNA methylation. Evidence from cell and animal models has indicated that m.1555A>G may make the 12S rRNA a better substrate for m⁶₂A methylation by the mitochondrial transcription factor B1 (TFB1M) enzyme, resulting in a higher ratio of methylated: unmethylated 12S rRNA transcripts in the mitochondrion, so called ‘hypermethylation’. Hypermethylation is thought to trigger a cascade of events, which results in mitochondrial dysfunction and hearing loss. Using a novel fluorescent method, suitable for paediatric patients, 12S m⁶₂A rRNA methylation was investigated in primary and non-primary cell-derived RNA samples from patients with m.1555A>G and controls. The data presented in this work indicates that previous findings may be an artefact of the experimental models used to study this hypothesis and 12S m⁶₂A rRNA methylation is unlikely to be a pathogenic mechanism. Aminoglycoside-induced reactive oxygen species (ROS) production is reported to play a key role in cochlear cell death. It is hypothesised that increased mitochondrial mRNA misreading disrupts oxidative phosphorylation, which leads to increased ROS. Using a multiplex flow cytometry-based method to measure mitochondrial superoxide, the response of cells from cases with m.1555A>G and controls to aminoglycoside treatment was examined. The data indicates that the control response to aminoglycoside treatment is variable. Exploring this variation will be crucial if the effects of antibiotic-induced changes in mitochondrial mRNA misreading are to be investigated and the cellular events that ensue to be targeted for treatment.

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