Energy production & mitochondrial DNA function in normal and disease states

Lead Research Organisation: MRC National Inst for Medical Research

Abstract

Food is utilised by the compartment of the cell called mitochondria to generate the bulk of the energy required by the human body. A small piece of DNA located in mitochondria makes an essential contribution to energy production. Mutations in mitochondrial DNA cause a wide range of diseases, including neurodegeneration, diabetes mellitus and cardiac dysfunction, which are important causes of morbidity. We are studying how this essential molecule is copied and inherited in order to design drug or molecular therapies for mitochondrial diseases.

Technical Summary

The aim of the group is to understand all aspects of mitochondrial DNA metabolism, in particular the parameters that determine fixation of pathological mitochondrial DNA variants in humans. Pathological mitochondrial DNA variants are implicated in a wide-range of human diseases and the accumulation of mutations in mitochondrial DNA has been linked to ageing. Our aim includes elucidation of the mechanism and machinery of mitochondrial DNA replication and maintenance, and identification of the factors influencing segregation of different mitochondrial DNA genotypes. DNA replication mechanisms are being studied using two-dimensional agarose gel electrophoresis in conjunction with atomic force microscopy and other techniques. Two schemes are to be employed to dissect the machinery of mitochondrial DNA replication; analytical protein chemistry of purified mitochondria and a combined reverse genetics and bioinformatics approach. Identification of factors that regulate replication and segregation of normal and mutant mitochondrial DNA offers a means to manipulate mutant load, initially in cell culture and subsequently in animal models of mitochondrial disease. Ultimately the aim is to restrict the propagation of mutant mitochondrial DNA in specific tissues of patients with mitochondrial disease, and potentially slow the natural process of ageing in normal individuals.

Publications

10 25 50
 
Description HEFA Oocyte Mitochondrial Transfer
Geographic Reach National 
Policy Influence Type Participation in a national consultation
Impact I provided expert advice on the safety of the proposed startegy to replace the nucleus of a healthy oocyte with the nucleues of a mother with a mitochondrial DNA disorder, as far as possible leaving behind the defective mitochondria.
 
Title affinity purification 
Description Various forms of affinity purificaiton have been devised over the years. We adopted the StrepII motif as it had been used successfully for purifcation of proteins from bacteria. We have demonstarted that it is an extremely effective means of purifying tagged human proteins from cultured cells. 
Type Of Material Biological samples 
Year Produced 2010 
Provided To Others? Yes  
Impact Using this procedure we identified the accessory subunit of the mitochondrial RNA polymerase that is essentail for the synthesis of polycistronic transcripts in mitochondria (without which cells cannot respire. Second, we showed that C4orf14 is a ribosomal assembly factor for the small subunit of the mitochondrial ribosome. The method underpinned our study that showed the mitochondrial nucleoid and protein synthesis machinery are couple din mitochondria. RThis fiding has major implications for the expression of mitochondrial DNA and must be considered in the context of mitochondrial versus cytosolic protein synthesis and longevity. 
 
Title in organello rep 
Description A method to measure DNA synthesis in isolated mitochondria 
Type Of Material Technology assay or reagent 
Provided To Others? No  
Impact PMID: 23595151 
 
Description Mitochondrial DNA Organisation 
Organisation Radboud University Nijmegen
Country Netherlands 
Sector Academic/University 
PI Contribution Intellectual, practical and financial
Collaborator Contribution Reagents (cell lines, constructs), Intellectual
Impact Three joint publications, to date.
 
Description Mitochondrial DNA Segregation 
Organisation University of Padova
Country Italy 
Sector Academic/University 
PI Contribution Intellectual and minor practical contributions made to Dr Vergani's research programme.
Collaborator Contribution Helped to identify factors underpinning biased segregation of mutant and wild-type mitochondrial DNA
Impact Demonstrated the effect of the mitochondrial fission apparatus on the segregation of mutant and wild-type mitochondrial DNA, with implications for the associated human disease. Paper published in Human Molecular Genetics Journal in 2009
 
Description Mitochondrial DNA replication Mechanisms 
Organisation Autonomous University of Madrid
Department Centre for Molecular Biology Severo Ochoa
Country Spain 
Sector Academic/University 
PI Contribution Intellectual, practical and financial
Collaborator Contribution Intellectual
Impact numerous publications on the topic
 
Description Mitochondrial DNA replication Mechanisms 
Organisation National Institutes of Health (NIH)
Country United States 
Sector Public 
PI Contribution Intellectual, practical and financial
Collaborator Contribution Intellectual
Impact numerous publications on the topic
 
Description Mitochondrial DNA replication Mechanisms 
Organisation Tampere University of Technology
Country Finland 
Sector Academic/University 
PI Contribution Intellectual, practical and financial
Collaborator Contribution Intellectual
Impact numerous publications on the topic
 
Description RNase H1 
Organisation National Institutes of Health (NIH)
Country United States 
Sector Public 
PI Contribution We helped define the sub-cellular compartments to which RNaseH1 is trafficked We are characterizing the effects on mitochondrial DNA replication on the loss of RNase H1, using a cell line developed by Dr Crouch
Collaborator Contribution They define the sub-cellular compartments to which RNaseH1 is trafficked RNase H1 ablated cell line.
Impact PMID: 20823270 PMID: 20184890
Start Year 2008