Effects of alternative oxidase expression in mitochondrial DNA helicase mutants of Drosophila melanogaster

Author(s)

Ana Paula Campos Rodrigues

• Document type: Master's Dissertation
• Institution: Universidade Estadual Paulista (Unesp)
• Defense date: 2017-07-25
• Advisor: Marcos Tulio de Oliveira

Abstract

Mutations in the human nuclear gene C10orf2, which encodes the mitochondrial replicative helicase Twinkle, are associated with cases of autosomal dominant Progressive External Ophthalmoplegia (adPEO) with multiple deletions in the mitochondrial DNA (mtDNA), and other diseases in humans. Two mutations analogous to those found in human patients (W441C and A442P) and an active site mutation (K388A) were modeled in Drosophila melanogaster. The expression of the mutant enzymes led to defects in mtDNA replication, impairment of mitochondrial oxidative phosphorylation (OXPHOS), and developmental disorders in flies. Transgenic expression of the mitochondrial alternative oxidase (AOX) has been shown to be efficient in combatting mitochondrial dysfunctions caused by genetic defects, mainly due to the bypass of complex III and IV. Thus, our aim was to test whether AOX expression can rescue the deleterious developmental effects caused by Twinkle mutations in D. melanogaster. Phenotypic data has shown that AOX expression in Twinkle mutants K388A and A422P was unable to prevent the lethal phenotype at the larval or pupal stage, and AOX was not effective against the mtDNA depletion of these flies. The W441C mutants, in the presence or absence of AOX, did not show developmental problems, nor changes in mtDNA copy number. Lines overexpressing the wild-type Twinkle (WT) and AOX had normal viability, in addition to an increase in mtDNA copy number in adults, not attributed to AOX. By measuring OXPHOS activity in tissues unrelated to metamorphosis, we detected no dysfunctions associated with the expression of mutant or WT Twinkle, nor a significant alteration by the presence of AOX. Our data indicates that the use of AOX as a gene therapy strategy to help cases of Twinkle mutations has limitations, and that perhaps the role of the mitochondrial replicative helicase in animal physiology is much more complex than previously thought. (AU)

• FAPESP's process: 15/14547-7 - Effects of AOX expression in mitochondrial DNA helicase mutants of Drosophila melanogaster
• Grantee: Ana Paula Campos Rodrigues
• Support Opportunities: Scholarships in Brazil - Master