Functional characterisation of coq8 in Drosophila : a thesis presented in partial fulfilment of the requirements for the degree of Masters of Science in Genetics, Massey University

Abstract

With the increasing number of novel mutations being discovered by whole genome and whole exome sequencing, functional studies are increasingly required to determine whether specific mutations are responsible for the disease phenotypes. 𝘋𝘳𝘰𝘴𝘰𝘱𝘩𝘪𝘭𝘢, with its vast set of genetic and molecular tools as well as robust behavioural assays, is an ideal model for functional characterisation.

Coenzyme Q biosynthesis is highly conserved from yeast to humans and involves a number of genes in the enzymatic pathway including COQ8A. The role of COQ8A in CoQ biosynthesis is not clear. However, mutations in COQ8A have been associated with autosomal recessive cerebellar ataxia, which is characterised by gait ataxia, cerebellar degeneration and CoQ₁₀ deficiency. This project aimed to characterise the phenotypes resulting from the reduction of 𝘤𝘰𝘲8 expression (the 𝘋𝘳𝘰𝘴𝘰𝘱𝘩𝘪𝘭𝘢 homologue of COQ8A) to develop a model of 𝘤𝘰𝘲8 deficiency that could be used to characterise COQ8A mutations functionally.

RNAi knockdown of 𝘤𝘰𝘲8 resulted in severe developmental delay, larval lethality, locomotor impairment, a decrease in ATP production, as well as developmental deficits and neurodegeneration in the Drosophila eye. Reintroduction of wild-type 𝘋𝘳𝘰𝘴𝘰𝘱𝘩𝘪𝘭𝘢 coq8 partially rescued the larval lethality, restored locomotor function and also primarily rescued the necrotic phenotype in the eye. This model could, therefore, be used to determine whether a specific mutation impaired function, such that it would not rescue the deficiency. As a proof-of-principle, two mutant variants of 𝘤𝘰𝘲8, I295P and L520*, which were modelled on the human COQ8A mutations L277P and c.1506+1G>A (which results in a truncated protein) did not rescue the 𝘤𝘰𝘲8 deficiency, indicating that they disrupted normal 𝘤𝘰𝘲8 function. However, the reintroduction of human COQ8A did not restore function but instead exacerbated the necrotic and neurodegenerative phenotype in the eye suggesting that it may be impairing the mitochondrial function of wild-type 𝘤𝘰𝘲8.

𝘋𝘳𝘰𝘴𝘰𝘱𝘩𝘪𝘭𝘢 provides the means to characterise disease-causing genetic mutations functionally. Here we have developed a model that can be used to study the role of 𝘤𝘰𝘲8 in 𝘋𝘳𝘰𝘴𝘰𝘱𝘩𝘪𝘭𝘢 and have found that 𝘋𝘳𝘰𝘴𝘰𝘱𝘩𝘪𝘭𝘢 𝘤𝘰𝘲8 and human COQ8A differ in function.