Mitochondrial ROS mapping and control with sub-organellar resolution

Lead Research Organisation: University of Glasgow
Department Name: College of Medical, Veterinary &Life Sci

Abstract

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.

Technical Summary

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.

Planned Impact

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.
 
Title Mitochondria and Us Video 2021 
Description Video (15 min) including interviews with PI and lab members, recordings from the webinar, animation film provided by the Lily Foundation and photographs and videos taken at the University of Glasgow (external shooting and shooting in the lab) 
Type Of Art Film/Video/Animation 
Year Produced 2021 
Impact The video is used as a resource for outreach activity at the University of Glasgow, at the RSE and the Lily Foundation 
 
Title Video on Crossover Workshop with Glasgow School of Art Innovation School 
Description A video was generated that capctured the views, feedback, ideas that were generated during the Crossover Workshop between Design and Biochemical Sciences 
Type Of Art Film/Video/Animation 
Year Produced 2020 
Impact Access to wider audience including artists and designers and generation of interest in working at the interface between design and Biochemical Sciences to develop new public engagement activities with new routes to impact 
 
Description We have synthesized a series of small chemical molecules that can specifically detect distinct reactive oxygen species (ROS), which are damaging to the cell. It is important to understand where these species are made in the cell, and we have developed a series of protein constructs in specific sub-mitochondrial compartments that specifically attach to these probes. We will make use of these constructs in healthy and compromised or stressed cells to detect for the first time the ROS distribution and effects in damaged mitochondria.
Exploitation Route still work under way -
The new probes and our technology can be used in a whole range of problems (ageing, common diseases like neurodegenerative diseases and cancer) to link the ROS production and their effect on mitochondria fitness and cell physiology.
Sectors Chemicals,Education,Healthcare,Manufacturing, including Industrial Biotechology,Pharmaceuticals and Medical Biotechnology

 
Description Our research findings so far highlighted a new level of complexity in the internal architecture of mitochondria and how this may be linked to ROS production - We have highlighted these issues as underpinning fundamental research in mitochondria in platforms where advances in mitochodrial research are relevant (patient groups, healthcare industry)
First Year Of Impact 2021
Sector Chemicals,Education,Healthcare,Manufacturing, including Industrial Biotechology,Pharmaceuticals and Medical Biotechnology
Impact Types Societal,Economic

 
Description Engagement with Lily Foundation mitochondrial patient charity
Geographic Reach National 
Policy Influence Type Contribution to new or Improved professional practice
Impact The Lily foundation is the largest mitochondrial patient charity in the UK. I have advised them on issues regarding new ways to simplify scientific research findings and make them more accessible to their members and more broadly the public.
 
Description Engagement with MyMitomission patient charity
Geographic Reach National 
Policy Influence Type Membership of a guideline committee
 
Description CiC-A first-in-class peptide-based platform delivery technology to modulate mitochondria function in disease
Amount £122,738 (GBP)
Organisation Medical Research Council (MRC) 
Sector Public
Country United Kingdom
Start 04/2021 
End 06/2022
 
Description IAA joint BBSRC-EPSRC award:Mitotargin: A novel peptide tool for external payload delivery to mitochondria in cells
Amount £33,844 (GBP)
Organisation Biotechnology and Biological Sciences Research Council (BBSRC) 
Sector Public
Country United Kingdom
Start 06/2019 
End 01/2020
 
Description Tokatlidis - Inaugural Wellcome Early Concept Development Fund
Amount £32,318 (GBP)
Organisation Wellcome Trust 
Sector Charity/Non Profit
Country United Kingdom
Start 09/2020 
End 03/2021
 
Description Wellcome Trust Translational Partnership - Industry Champion - Keith Wreggett for Mitotargin
Amount £13,200 (GBP)
Organisation Wellcome Trust 
Sector Charity/Non Profit
Country United Kingdom
Start 11/2021 
End 07/2022
 
Title Fluorescence assay for monitoring peptide targeting into isolated mitochondria 
Description We have established a new method for monitoring uptake of targeting peptides to isolated mitochondria, using a fluorescently labelled synthetically made peptide. The assay is used to assess the capacity of a peptide to be targeted and internalised into the mitochondrial intermembrane space (IMS). It has been validated for peptides that follow distinct import routes into the IMS. Specificity has been tested using mitochondria devoid of key protein import components as controls. 
Type Of Material Technology assay or reagent 
Year Produced 2019 
Provided To Others? No  
Impact Could be used for testing the mitochondrial targeting capacity of any synthetic peptide (libraries) with a fluorescent moiety tagged to the peptide 
 
Title SNAP and HALO tagged cells for fluorescent probe detection of ROS with sub-mitochondrial resolution 
Description We have generated SNAP- and Halo tagged versions for cells expressing the tags in all submitochondrial locations. These can be used to label (using appropriate fluorescent ligand probes selective for SNAP or Halo proteins) in specific sub-compartments ROS species (hydogen peroxide and superoxide) 
Type Of Material Technology assay or reagent 
Year Produced 2021 
Provided To Others? No  
Impact too early for impact . Expected to generate impact after publication of technology 
 
Title peptide based technology for delivering payload to mammalian cells 
Description developed a platform technology for delivering payload (small molecules, peptides and small proteins) to mitochondria in mammalian cells as a way to modulate their function. Applications range from improving mitochondrial function in cases of primary mitochondria diseases and common conditions (neurodegeneration, diabetes) where mitochondria is an underpinning problem to blocking mitochondria function as a means of cell killing of cancer cells. 
Type Of Material Technology assay or reagent 
Year Produced 2020 
Provided To Others? No  
Impact IP rights generated - patent filed Initiated discussion with Venture capital companies interested in the technology New funding obtained (MRC Proximity to discovery award, BBSRC-EPSRC Impact accelerator award, Wellcome Trust Early stage discovery award) Market research outsourced to company and completed 
 
Description Collaboration with Dr Brian Smith (UofG-IMCSB) on NMR analysis on the initial binding to mitochondrial receptors of the N18 peptide of Gpx3 which is synthesized in response to oxidative stress 
Organisation University of Glasgow
Department Institute of Molecular Cell and Systems Biology
Country United Kingdom 
Sector Academic/University 
PI Contribution We have started collaborative experiments with Dr Brian Smith using NMR analysis to determine the mode of biding (with high resolution) of the N18 extended peptide that guides Gpx3 to the surface of mitochondria and interacts with the receptor Tom20. My team is purifying the proteins, and does the labeling (N15) for NMR experiments.
Collaborator Contribution Dr Brian Smith is taking the NMR spectra of the proteins in isolation or in complex with each other to analyse their interaction
Impact Spectra of Gpx3 and N18Gpx3
Start Year 2020
 
Description NMR analysis of MIa40 and Gpx3 interactions with CERM-Florence Italy 
Organisation University of Florence
Department Magnetic Resonance Centre
Country Italy 
Sector Academic/University 
PI Contribution purification and labeling of proteins, development of protocols for interactions
Collaborator Contribution NMR analysis of isolated proteins and of their complexes
Impact multidisciplinary collaboration with chemists and spectroscopists
Start Year 2020
 
Title Targeting peptide 
Description novel type of targeting peptide for mitochondria 
IP Reference  
Protection Patent application published
Year Protection Granted 2021
Licensed No
Impact in discussions to set up a spinout company
 
Description Crossover Workshop on Mitochondria and Us 
Form Of Engagement Activity Participation in an activity, workshop or similar
Part Of Official Scheme? No
Geographic Reach National
Primary Audience Other audiences
Results and Impact The Mitochondria and Us workshop (25th February 2021) was done in collaboration with Enio Caccavale and Michael Johnson from the GSA and with support fomr the Biochemical Society and the Young Academy of Scotland (RSE).
This workshop has invited a broad range of disciplinary expertise and interests to explore how to co-create stories at the societal, experiential and biochemical levels to provide greater understanding of people's experiences living with and working with mitochondrial diseases.
Based on an image and 100 words summary in response to the question: How could your expertise contribute to a societal understanding of rare and invisible diseases (such as mitochondrial diseases)? the participants discussed in small groups a number of issues pertaining to mito diseases.
The responses and contributions of the disciplines and motivations being brought together established crossover connections in the workshop The whole grouped discussed on innovative ways of engagement during the workshop activities. The outcome was thought by participants to shape novel, stimulating and important dialogues to achieve a better awareness among the different disciplines and with the general public.
Year(s) Of Engagement Activity 2021
URL https://www.gsainnovationschool.com/post/crossover
 
Description Organized Interdisciplinary webinar on Mitochondria and Us with RSE support for mitochondrial awareness week 2020 
Form Of Engagement Activity Participation in an activity, workshop or similar
Part Of Official Scheme? No
Geographic Reach International
Primary Audience Professional Practitioners
Results and Impact A webinar event bringing together experts from several disciplines (science fields incl biology, chemistry, medicine, physics data science, ethics, designers, social science, patient groups) to discuss research advances and impact on society of mitochondria dysfunction. The was more than 350 participants from global locations (Europe, Americas, Asia, Australia, Africa). I chaired and organized the event, with co-organisers from the Glasgow School of Art, and support from the Royal Society of Edinburgh and the Lily Foundation (the largest UK mitochondria disease patient organization). There were 2 one-hour sessions with short talks (12 speakers in total) representing all disciplines , ECRs, and the organization of women working in mitochondria. There were two Q and A session with questions asked by the audience and a final panel discussion at the end.
Year(s) Of Engagement Activity 2020
URL https://www.rse.org.uk/event/mitochondria-and-us/
 
Description Press release on Mitochondria and Us webinar 
Form Of Engagement Activity A press release, press conference or response to a media enquiry/interview
Part Of Official Scheme? No
Geographic Reach International
Primary Audience Media (as a channel to the public)
Results and Impact Press release generated for the Mitochondria and Us webinar. Distributed to media via the Royal Society of Edinburgh comms channels
Year(s) Of Engagement Activity 2020
URL https://www.rse.org.uk/energising-cells-zooming-cells-powerhouse-scotland/