Mitochondrial DNA replication and disease
A variety of human diseases, ranging from devastating conditions of infancy through to degenerative disorders seen mainly in old age, are associated with genetic lesions of mitochondrial DNA. In order to understand how mtDNA maintenance shapes cell physiology, and elucidate how it can go wrong in disease and ageing, we are studying the fundamental mechanisms of DNA replication in model organisms, using a combination of genetic and biochemical approaches.
• Our recent findings support the so-called bootlace model of mtDNA replication, in which preformed transcripts are incorporated into replication intermediates, to create a provisional lagging-strand.
• Consistent with this model, but with slightly different kinetics than in mammalian systems, mtDNA replication in Drosophila depends upon the stoichiometrically appropriate binding of proteins of the mitochondrial transcription termination (MTERF) family.
• RNaseH1 is required for mtDNA maintenance in both mammals and flies. In mammals, its absence results in failure to remove the replication primers, which blocks future replication rounds. Current work in Drosophila aims to determine whether RNaseH1 also plays additional roles in mtDNA replication.
• In C. elegans, where mtDNA replication occurs only in the female germline, we find a signature of rolling-circle replication.
• A fully functional ATP synthase complex is required for mtDNA maintenance in Drosophila
Nuclear-mitochondrial interactions in Drosophila
We use various Drosophila mutants with defects in mitochondrial function to analyse the contributions of nuclear and mitochondrial genotype, as well as environmental factors such as diet and exposure to antibiotics, to organismal phenotype. Much of our attention has been focusSed on the tko25t strain, which carries a point mutation in the gene for mitoribosomal protein S12, and is a useful model for studying human mitochondrial disease. We are also studying the roles in animal development of global regulators of mitochondrial function, and the physiological effects of respiratory chain dysfunction in the nervous system, using flies as a model system.
• Altered gene expression and metabolite profiles in tko25t indicate reliance on glycolysis, but concomitant build-up of potentially toxic intermediates such as pyruvate and lactate, as well as depletion of ATP and profound redox disturbances
• The tko25t mutant phenotype can be partially alleviated by specific mitochondrial backgrounds in cybrid flies, and is also be susceptible to manipulations of the nuclear genome.
• The sesB1 mutant, affecting the major isoform of the adenine nucleotide translocase, shares many features with tko25t. Its phenotype is also subject to modification by genetic background, but in subtly different ways.
• On-going work on the contribution of mtDNA genotype to the phenotype of tko25t is a joint project with Finland Distinguished Professor Laurie Kaguni (Tampere, Michigan State).
Alternative respiratory chain enzymes: a possible therapy for mitochondrial disorders?
The genomes of plants, fungi and many microbes, as well as some primitive animal phyla, contain genes for alternative mitochondrial respiratory chain enzymes, which buffer a host of redox and bioenergetic stresses similar to those encountered in humans under pathological conditions. The genes for these alternative enzymes are absent from humans and other complex animals. However, we reasoned that their introduction may alleviate many of the physiological defects associated with mitochondrial disease. We have transferred the relevant genes from the tunicate Ciona, as well as from fungi, into human cells and model organisms, creating proof of concept for such protection. Our current research focuses on ascertaining the potential of these enzymes in therapy, both by exploring what specific pathologies they can prevent, as well as testing out ways to safely introduce them in a therapeutically effective form to humans. In addition we are studying the natural biology of the alternative enzymes in Ciona, to understand better how they can be most effectively used in humans.
• The alternative oxidase (AOX) and NADH dehydrogenase (NDX) from Ciona can be expressed in model organisms in all tissues and throughout the life cycle without any significant deleterious effects.
• AOX and NDX confer resistance against respiratory poisons (e.g. cyanide, antimycin, rotenone) on both cells and whole organisms
• AOX protects cells and tissues form the effects of chronic exposure to mitochondrial toxins, such as found in cigarette smoke
• NDX and AOX can protect Drosophila from lethality and other severe phenotypes caused by genetic ablation of respiratory chain complexes I and IV
• AOX also protects against pathological insults connected to oxidative stress