Mitochondrial ROS mapping and control with sub-organellar resolution

This project aims to advance our understanding of how mitochondria are able to retain their function under severe stress. Mitochondria are fundamental components of animal cells that are essential for proper cell metabolism and physiology. Mitochondrial dysfunction is linked to ageing, and to numerous human pathologies and so the maintenance of their normal function is very important. Mitochondria produce small molecules called reactive oxygen species (ROS) which are important for signalling in the cell but at high levels become highly damaging. The project will elucidate where within mitochondria ROS are produced, and how their levels are maintained. This will in turn allow us to understand better their localised effects as they are dictated by the internal mitochondrial architecture as a first step to develop efficient and targeted strategies to control them. This is absolutely critical because maintaining a fine balance of their levels makes all the difference for ROS being beneficial as signalling molecules in low levels or reverting to damaging agents for the cell at high levels.
We will synthesise a series of small chemical molecules that can specifically detect distinct ROS and localise them in specific sub-mitochondrial compartments by targeting to these compartments selective protein tags that specifically attach to these probes. Under healthy conditions the import of these protein tags is powered by the mitochondrial transmembrane energy gradient. We have discovered that one antioxidant protein is imported by a system that does not require this energy gradient, which is remarkable and just as well since that gradient is compromised under severe redox stress conditions. We will make use of our knowledge of this new import pathway in compromised mitochondria to address for the first time the ROS distribution and effects in such damaged mitochondria.
Our findings offer the first opportunity to explore the mechanisms of a previously elusive ROS distribution with unprecedented sub-mitochondrial resolution. This is critical to effectively defend cells against deleterious oxidative stress. Our interdisciplinary approach outlined in this project will further our understanding of mitochondrial targeting and cell stress mechanisms and it is likely to provide a novel paradigm for understanding the coordination of oxidative stress signalling in eukaryotic cells.

This proposal aims to elucidate the site of production and levels of different mitochondrial ROS with sub-mitochondrial resolution. Estimation of ROS levels has been so far based on average values neglecting the internal mitochondria architecture. This will in turn allow us to understand better their localised effects and how antioxidant mechanisms can regulate them. This is absolutely critical because maintaining a fine balance of their levels makes all the difference for ROS being beneficial for signalling in low levels but reverting to damaging agents at high levels. We will focus on superoxide and hydrogen peroxide capitalising on two important advances made by the co-applicants. First, the discovery of new protein targeting pathways (Tokatlidis lab), one of them independent of the transmembrane potential, which can allow precise protein targeting within mitochondria sub-compartments (bulk intermembrane space, crista lumen, outer surface of inner membrane, matrix surface of inner membrane and bulk matrix). Second, the generation and validation of new fluorescence ROS sensors with improved specificity and detection (Hartley lab). SNAP-tag and HALO-tag label proteins will be localised to each mitochondrial sub-compartment by fusion to appropriate targeting peptides. Subsequently, fluorescent sensors specific for superoxide and hydrogen peroxide will be fused to ligands that attach covalently to the tag and these sensor-bearing ligands will be added exogenously to map the level and distribution of the ROS species within the sub-mitochondrial compartments (objective-1). We will then investigate how intra-mitochondrial ROS change upon cellular redox stress perturbations (objective-2), and establish a link between changes in ROS levels and alterations of internal mitochondrial architecture and antioxidant protein targeting (objective-3). Understanding mechanisms that can potentially protect mitochondria from irreversible oxidative damage are very important.

This proposal has the potential to impact on academic and industrial researchers in a wide range of disciplines, but also the UK economy by the generation of marketable compounds. It will also impact on society by guiding novel strategies for therapy and diagnosis of mitochondrial dysfunction. Mitochondrial function is central to life and their dysfunction is involved in a wide range of neurodegenerative and cardiovascular diseases, inflammation and the process of ageing. Mapping ROS with new probes will solve the mystery of redox regulation, providing a so far elusive yet critically needed sub-organellar resolution. This will impact on fundamental knowledge of how cells and mitochondria remain healthy under oxidative stress. By addressing such critical bioscience questions for cell and chemical biologists it will spur translational applications engaging biomedical and clinical scientists. The main routes to scientific impact will be through research publications and reviews in high impact journals, lectures at national and international meetings, at universities and to pharmaceutical companies. Two PDRAs will be trained in cutting edge research techniques, and will have their communication skills developed by presenting their work at national and international meetings. The PDRAs will be working closely with the collaborating team, which will be operating in a distinctly different discipline, and will be helped to develop skills for interdisciplinary communication. The work will generate new molecular probes that will be widely used in biomedical science. Sufficient quantities of the molecular probes will be made during the course of the study to supply other labs. The probes will be made immediately available to collaborating labs in cardiovascular research, redox signaling and mitochondrial research (see letters of support) and to other labs upon request. Hartley has a track record in engaging with commercial suppliers and will bring the new molecular probes rapidly to market for both scientific and economic benefit. The detailed understanding of ROS in the cell's powerhouse and metabolic signaling hub, the mitochondria, will suggest new strategies for intervention in the wide range of diseases where mitochondria play a role (recently reviewied by Murphy and Hartley). These strategies should will constitute new intellectual property. Both Tokatlidis and Hartley are experienced in translating discoveries through patenting. We will exploit opportunities for translation from the BBSRC (e.g. follow-on funding) and other sources such as Scottish Enterprise and through direct funding from industry should IP arise. Both teams also have strong track records in public and media engagement. The proposal schedules showcase activities each year in the Hunterian museum or in the Glasgow Science Centre. These will be complemented by a wide range of other engagement activities including those where we have a track record: lectures to High Schools, hosting High School pupils, Twitter, our websites, Science Slam and other events, and giving interviews to local/national TV and radio. We will evaluate the progress towards Impact at six-monthly intervals. This will be done together with the Business Development Office and will be based around milestones. Three are short-term. Milestone 1: Dissemination of results to the academic community (high-impact journals, national/international conference talks). Milestone 2: Expand existing networks and create new ones (such as ITN and Leverhulme Trust International Network). Milestone 3: Establish two to three STSMs within the COST networks during the project. Milestone 4 is short-medium term: Make our new molecular probes available to other academic and industrial groups to enable further research in targeting to mitochondria. Milestone 5 is long-term: Novel diagnostics and therapeutics in mitochondrial medicine informed by this research leads to improved quality of life for affected patients.