Regulation of active/deactive transition of mitochondrial complex I in health and pathology

Lead Research Organisation: Queen's University of Belfast
Department Name: Sch of Biological Sciences

Abstract

Currently in the UK every six minutes a person dies of heart disease and every five minutes another person dies of stroke. The impact of stroke on the UK economy is about £7.0 billion in NHS, post care costs and benefit payments. Our project aims to improve the situation with these pathologies.
The reason behind death or damage during heart failure and stroke episodes is a lack of oxygen or "hypoxia" in the tissue. Hypoxia causes this damage because it results in impairment of energy balance in cells. Production of energy in the body is provided by the action of the respiratory chain in small specialised structures known as mitochondria - "power stations" of the cell. They are the main oxygen consumers and 99% of the oxygen we breathe is used by mitochondria. They generate energy needed for the body such as heart beating, brain activity, conduction of nerve impulses, DNA biosynthesis and cell growth. If oxygen is present, our cells "respire" and mitochondria produce energy. Without oxygen mitochondria cannot generate energy, and moreover, when oxygen supply is restored mitochondria generate detrimental free radicals. These radicals damage all the surrounding structures including mitochondrial proteins, DNA and membranes.
An enzyme called mitochondrial Complex I is the key protein in the mitochondria since it starts a sequence of reactions that eventually produces energy. It is a huge enzyme and it is still not clear how it works or how cells regulate it. Defects in Complex I are associated with many human disorders such as Parkinson's disease, muscle problems and stroke-like episodes in children. Despite decades of studies, we still do not know exactly how Complex I works and we are still unable to prevent damage to mitochondria during stroke or heart failure. Recently we found that there are two different forms of Complex I present in tissues: either an active or a sleeping, dormant form. We found that the dormant form is particularly susceptible to modification b several biological molecules including free radicals. Depending on physiological conditions, it may cause either protection of the tissue during hypoxia or make hypoxic damage worse. This finding opened a completely new area of research on a link between Complex I and oxygen. Complex I does not require oxygen for functioning but surprisingly it can be regulated by oxygen concentration in the cell. We want to understand this interesting phenomenon and identify the conditions in which natural, protective de-activation of Complex I may be disadvantageous.
We also plan to investigate special molecules from the cellular membrane called fatty acids because our preliminary data show that they strongly affect the active/dormant state of the enzyme. Fatty acids are long water-insoluble molecules that are part of fat-containing food. Therefore their concentration in our body is moderated by diet and is altered in obesity.
We are going to use modern proteomic techniques to carry out structural studies of Complex I at the biochemical level in intact mitochondria, cells and tissues. We will also use advanced non-invasive cellular respirometry techniques for measuring how cells consume oxygen. Also we will be using fluorescent confocal microscopy, a special technique to look at the different parameters in a single alive cell. Our research will help to understand the mechanisms of changes in mitochondria and cells after stroke or infarction and outline the way to ameliorate the damage after the episode has happened. In addition it would help to develop approaches for early diagnosis, and prevention of the initial stages of neurodegenerative conditions such as Parkinson's disease. It will also lead to the development of new drugs that can selectively regulate Complex I activity in order to diminish hypoxic injury in tissues like heart and muscle in disease and to control age-related damage of nerve cells in the brain.

Technical Summary

In our previous work we characterised two interconvertible subpopulations of Complex I in vitro and in vivo: catalytically active (A) or de-active (D). Our recent findings indicate that we have uncovered a key mitochondrial mechanism directly involved into these processes. However the details of the dynamics of conversion between the two forms of the enzyme in situ are still not clear. We will study the regulation of the A/D transition of mitochondrial Complex I and characterise the effect of several endogenous effectors on ischaemia/reperfusion (I/R) damage. In the proposed project we will:
RQ1. Characterise the interaction of nitrosothiols, peroxynitrite and ROS with both forms of Complex I from brain mitochondria
RQ2. Investigate the effect of free fatty acids and their metabolites on the ability of the enzyme to undergo A to D and D to A conversion
RQ3. Characterise the A/D transition of Complex I from mitochondria isolated from obese animals
RQ4. Identify the role of anaerobic NADH:fumarate reductase activity catalysed by Complexes I and II together in modulating of A/D equilibrium in situ.
This work will include a detailed analysis of the A/D transition in brain mitochondria, neuronal and cardiac cell lines and tissues ex vivo. We will assess the effect of several types of fatty acid metabolite. After identification, effectors with the most manifest action will be used to test the effect of metabolites on cell cultures of neurones in an I/R model.
The analysis would also consist of monitoring enzymatic activities, A/D ratio and ROS production during fast reoxygenation of hypoxic cells. Together with other parameters such as respiration, degree of oxidative damage and cell viability it would give us a reliable picture of how A/D equilibrium is regulated and what is happening with mitochondrial bioenergetics during I/R. Furthermore this will enable us to correlate the state of Complex I with the physiological state of the tissue before and after I/R damage

Planned Impact

Stroke and cardiovascular diseases are highly prevalent in our society. In the UK, there are about 124,000 incidents of heart attack and around 152,000 strokes each year, resulting in over 131,000 deaths. Stroke costs the UK economy about £2.8 billion in direct costs to the NHS, £2.4 billion in post care costs and £1.8 billion in benefit payments. Patient recovery rate is not improving rapidly and on average stroke patients occupy around 20% of all acute hospital beds and 25% of long-term beds. Absence of oxygen and then reoxygenation are the main reasons for the tissue damage occurring in stroke and heart failure. What is needed is a more evidence-based and physiologically relevant understanding of the initial molecular mechanisms of damage development and more detailed insight into how tissues recover after the episode has occurred.
We aim to investigate these problems in several ways:
1. In the short term our work will be of interest to pharmaceutical and biotechnological industries, where there is currently great interest (and investment) in the development of pharmacological interventions and therapy for ischaemia/reperfusion and stroke.
2. Understand the mechanisms of post stroke changes in brain and find a way to ameliorate impairment after the episode has occurred.
3. Planned experiments using diet-induced obesity model will help to develop new types of more effective diets for people suffering from this disorder
Improved understanding of Complex I mechanisms and it's regulation in hypoxia are relevant to many other problems of fundamental and applied research including; nutritional science, sport specialists, fuel cell design for bioenergy production and transplantation studies. As such the potential societal and economic impacts of the research are broad.
1. As with much fundamental research our current project will benefit society in the long term by providing better comprehension of fundamental roles of mitochondria in cellular response to hypoxia
2ur research, if successful, would increase the rating of UK science worldwide, place it at an advantageous position and attract high quality scientists as well as lead to the generation of new intellectual property of commercial value.

These advances are realistic in the medium term, and build on our recent successes. What is needed at the moment is a systematic understanding of how the Complex I conformational change is regulated, the key natural effectors and the consequences of de-activation of the enzyme.

Publications

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Ana Stepanova (2018) Deactivation of Mitochondrial Complex I after Hypoxia-Ischemia in the Immature Brain in Journal of Cerebral Blood Flow & Metabolism

 
Title Mitochondrial Complex I Home Page support 
Description The original site has been established by Uli Brandt and Akemi Yagi in the '90s. The purpose of this site is to provide general information regarding complex I (NADH:quinone oxidoreductase) to researchers in the field of bioenergetics as well as to those who are simply curious about what this enzyme complex does 
Type Of Art Artefact (including digital) 
Year Produced 2016 
Impact The original site has been established by Uli Brandt and Akemi Yagi in the '90s. The purpose of this site is to provide general information regarding complex I (NADH:quinone oxidoreductase) to researchers in the field of bioenergetics as well as to those who are simply curious about what this enzyme complex does 
URL http://www.complexi.org/
 
Title Video of the A/D transition of mitochondrial Complex I 
Description Video of schematics of the A/D transition of mitochondrial Complex I 
Type Of Art Film/Video/Animation 
Year Produced 2016 
Impact Not known yet 
URL https://youtu.be/SMwQiVoLV1c
 
Description Cornell Medical College, Susan Vannucci 
Organisation Cornell University
Department Weill Cornell Medicine
Country United States 
Sector Academic/University 
PI Contribution Analysis of tissue samples obtained by using animal model. Characterisation of tissue injury in perinatal brain ischaemia
Collaborator Contribution Rat models of perinatal cerebral hypoxia-ischaemia
Impact Physiologically relevant model for studying involvement of mitochondria useful for amelioration of brain oxygen deprivation in newborn infants
Start Year 2014
 
Description Eugenia Trushina Mayo Clinic 
Organisation Mayo Clinic
Country United States 
Sector Charity/Non Profit 
PI Contribution Testing the effect of new neuroprotective drug on mitochondria
Collaborator Contribution Testing the effect of new neuroprotective drug on mitochondria
Impact Paper in preparation. Patent is pending.
Start Year 2016
 
Description MIke Murphy Cambridge MRC 
Organisation Medical Research Council (MRC)
Department MRC Mitochondrial Biology Unit
Country United Kingdom 
Sector Public 
PI Contribution Testing new MitoSNO compound in animal models
Collaborator Contribution Providing a new MItoSNO compound
Impact MitoSNO was found to me effective in neuroprotection
Start Year 2016
 
Description Maria E. Figueiredo-Pereira, Hunter College 
Organisation Hunter College
Country United States 
Sector Academic/University 
PI Contribution Cell models of proteosomal neuronal degeneration.
Collaborator Contribution Inhibitory analysis and biochemical analysis of respiratory chain enzymes
Impact Paper submitted to Journal of Biological Chemistry (JBC/2016/716035)
Start Year 2015
 
Description Nijmegen Center for Mitochondrial Disorders, Prof. Ulrich Brandt 
Organisation Nijmegen Centre for Mitochondrial Disorders (NCMD)
Country Netherlands 
Sector Academic/University 
PI Contribution Belfast - crosslinking of mitochondrial complex I and Blue Native Page and doubleSDS PAGE separation of subunits. Frankfurt - proteomics studies and identification of subunits.
Collaborator Contribution Identification of bovine Complex I subunits
Impact Map of 2D gels of separated subunits of bovine heart Complex I has been created
Start Year 2012
 
Description Pasquale Sansone Memorial Sloan-Kettering Cancer Center 
Organisation Memorial Sloan Kettering Cancer Center
Country United States 
Sector Academic/University 
PI Contribution Determination of dormant/awakening cells in the animal model of breast concer
Collaborator Contribution Determination of dormant/awakening cells in the animal model of breast concer
Impact Paper submitted to Nature Cell Biology
Start Year 2016
 
Description Annual Northern Ireland Bioscience Olympiad 
Form Of Engagement Activity A formal working group, expert panel or dialogue
Part Of Official Scheme? No
Geographic Reach International
Primary Audience Schools
Results and Impact At present, biological education in secondary schools makes biology a collection of facts and answering a question is usually limited to finding the right page in a textbook. The goal of the Bioscience Olympiad is to develop bioscience-oriented "academic thinking" and a creative approach to problem solving in school children. We aim to make the event not a contest of factual knowledge (with questions such as how many vertebras does a frog have or what is the name of an enzyme which cuts proteins) but one of intuitive and creative thinking. Answering the questions requires some basic textbook knowledge, but participants are invited to suggest as many "hypotheses" or "explanations" as they can, invent experiments to test their hypotheses and create a new classification of biological objects/processes (see Examples section).

In November 2010 I successfully launched the first pilot Olympiad was in the Medical Biology Building at Queen's University and a team from Antrim Grammar School won the Champions' cup. The contest consisted of a practical with theoretical components where participants had to do some experimental problem solving as well as answer questions.

Unlike many similar contests our Olympiad is not focused on "knowledge-testing," but rather the provision of alternative ways students to understanding natural sciences for the students. Another main goal is career-guidance for talented and capable school children and improvement of the quality of their education.

The winners of 2012 Olympiad were chosen to represent Republic of Ireland at the International Olympiad in Singapore.

Olympiad was sponsored in part by Biochemical Society and Irish Section of Biochemical Society.



Since 2010, participation in the Olympiad has increased from 7 (2010) to 24 (2014) secondary schools. More school students became aware of biochemical concepts in natural sciences.

We strive to bring science to life and show how science works, to emphasize problem solving aspects and critical thinking in bioscience and the understanding of main biological concepts.

I think we succeeded in developing teamwork and cooperative learning strategies among Northern Ireland secondary school students independent of their religious background.

After our events biological education is more interesting and more students will engage in natural science activities .

I believed that since 2010 we attracted more female students to professional and technical careers in natural sciences.
Year(s) Of Engagement Activity 2010,2011,2012,2013,2014
URL http://www.qub.ac.uk/Olympiad
 
Description Public understanding of Science lecture 
Form Of Engagement Activity A talk or presentation
Part Of Official Scheme? No
Type Of Presentation Keynote/Invited Speaker
Geographic Reach Local
Primary Audience Schools
Results and Impact 50 pupils from Downpatrick College attended my talk during Public Science week

http://go.qub.ac.uk/bioenergetics


Students visits and during so-called familarization week have been arranged
Year(s) Of Engagement Activity 2010,2011