Role of Mitochondrial Reactive Oxygen Species in Stress Adaptation during Ageing

Lead Research Organisation: Newcastle University
Department Name: Inst for Cell and Molecular Biosciences

Abstract

Nowadays, ageing is one of the main questions that modern biology needs to answer. We need to understand how and especially why we age to fully understand the process of evolution. In addition, a growing ageing population is one of the main problems in United Kingdom. The only way to alleviate the suffering caused by age-related degenerative disease (e.g. Alzheimer, Parkinson, cancer or diabetes) is to fully understand the underlying evolutionary forces, which drive ageing and design strategies to delay the ageing process. Mitochondria are the powerhouses of the cell generating most of the energy required for survival. These small cell factories deteriorate during ageing, failing to deliver the energy required for cellular maintenance. The reason why mitochondria fail is currently unknown, but it could be related with the way they produce energy. To operate, mitochondria use oxygen as final electron acceptor. Normally, this oxygen is safely managed by mitochondria being completely reduced to water with four electrons and two protons. However, in a minimal number of occasions oxygen is incompletely reduced (with less than four electrons) producing the so-called Reactive Oxygen Species (ROS) that can damage all cellular components.

The Mitochondrial Free Radical Theory of Ageing (MFRTA) was a popular theory to explain ageing in the past century. MFRTA proposes that ROS, produced as by-products of respiration, cause oxidative damage that accumulates and causes ageing. MFRTA is mainly supported by correlative data. Oxidative damage accumulates with age, and mtROS levels are altered in degenerative disease associated with ageing. However, direct experimental evidence fails to support MFRTA. Increasing mtROS does not shorten lifespan, and antioxidant supplementation has poor effects on health. It has been shown that mtROS are instrumental for cell differentiation, the immune response and stress adaptation. In conclusion, the contribution of mtROS to ageing is unclear. Because of the importance ROS have in pathological and non-pathological situations it is imperative to understand the physiological role they play in vivo.

In this proposal, we aim to understand in detail the role ROS play in normal physiology and in stress adaptation, particularly during ageing. Based on our preliminary results, we hypothesize that there are two different types of ROS populations. One population is good, and its generation is associated with the activation of mechanisms that clean up the cells. When these ROS are suppressed quality control mechanisms do not work properly and cellular homeostasis is lost. This would explain the negative consequences associated with supplementation or overexpression of antioxidants. The other population is deleterious, and it is produced only when mechanisms of mitochondrial quality control fail. These ROS are characterized by a very aggressive chemistry led by high levels of free iron and hydroxyl radicals.

Using the power of fruit fly genetics we will generate new transgenic models that will allow a precise manipulation of these two ROS populations in vivo. We will use this new technology to characterize the downstream physiological responses activated by ROS. We aim to find the exact pathways and genes that may be targeted by specific drugs or genetic interventions. These interventions should help to extend healthy lifespan. Since essential metabolic pathways are highly conserved during evolution, it is expected that similar strategies may be implemented in humans to delay ageing and prevent the onset of age-related diseases.

Technical Summary

During ageing, mitochondrial function is severely reduced. However, it is unknown why mitochondrial function deteriorates, and if this is a cause or a consequence of ageing. In many cell types, mitochondria are the main generators of Reactive Oxygen Species (ROS) and accumulation of oxidative damage has been postulated as the main cause of ageing in the past. However, mitochondrial ROS (mtROS) has two faces. One is negative, and decreases survival when ROS overcomes antioxidant defences. The other is positive, and participates in normal cellular signalling, extending lifespan when properly induced.

Our main hypothesis is that there are two main populations of mtROS. The first ROS population is generated when the ubiquinone (CoQ) pool becomes over-reduced. We propose that these ROS are implicated in the activation of quality control mechanisms that remove damaged molecules and organelles. This is the most prevalent ROS population in young cells. During ageing the accumulation of different types of damage blocks the transfer of electrons within respiratory complex I (CI). Under these conditions, electrons accumulate within CI stimulating the generation of superoxide that attacks the iron-sulphur clusters within CI. This causes the release of ferrous iron and the generation of hydroxyl radicals. These ROS are highly deleterious and are responsible for the negative effects associated with oxidative stress.

The main objective of this proposal is to understand by which mechanism(s) mtROS are physiologically produced, to delineate the role of mtROS in health and disease, and to find ways to manipulate mtROS in order to extend healthy lifespan. This main objective will be achieved through three specific aims: (i) manipulating the redox state of the CoQ pool, (ii) blocking the transfer of electrons within CI, and (iii) describing the physiological consequences produced as a consequence of (i) and (ii).

Planned Impact

Our main aim is to understand the role of mitochondrial Reactive Oxygen Species in cellular physiology. We will pay special attention to how ROS contribute to the decline in the capacity to confront stress that is observed during ageing. Three main social agents will benefit from our research: (i) academia, (ii) industry and (iii) society. Academia: ageing is one of the most fascinating questions in modern biology. Our project will increase knowledge at three fundamental levels molecular (mtROS), cellular (stress) and physiological (ageing). We will create new models to manipulate mtROS in vivo, we will describe specific mechanisms of stress adaptation during ageing and we will study new ways to extend lifespan manipulating evolutionary conserved signalling pathways. Scientists working on ageing and age-related disease will benefit from our research. Society: ageing is a priority of the research and social policies of both the United Kingdom and the European Union. It is imperative to find a way to extend healthy lifespan of the population and guarantee the long-term independence of senior citizens. Only basic research in ageing guarantees find new ways to delay and reverse ageing and prevent the onset of ageing-related diseases (Parkinson's and Alzheimer's disease, cancer, sarcopenia, etc). Therefore, our research will have a direct benefit improving health of the population. Additionally, publication of excellent research on ageing science catches mass media attention, and increases the public interest in healthier lifestyles. This alone could save millions in social security services. Industry: ageing is an emergent market for pharmaceutical companies. Our research will show new mechanisms to delay ageing and to identify specific targets in the form of signalling pathways and redox-regulated genes. Additionally, we will create new ageing and Parkinson's disease models to screen for new drugs and genetic interactions. This should be useful for pharmaceutical companies in order to design and test specific drugs against age-related diseases.

Publications

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Gubina N (2019) Essential Physiological Differences Characterize Short- and Long-Lived Strains of Drosophila melanogaster. in The journals of gerontology. Series A, Biological sciences and medical sciences

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Sanz A (2018) Editorial: Coenzyme Q Redox State and Cellular Homeostasis. in Frontiers in physiology

 
Description We have completed all the objectives proposed in specific aim 1, 2 and 3, finishing the project. Our most important findings and contributions are summarized below:
(1) We have generated different fly models where mitochondrial Reactive Oxygen Species (ROS) levels are controlled by modulating the redox state of the coenzyme Q pool (CoQ). These models take advantage of alternative respiratory enzymes, genetic manipulation of the levels of respiratory complexes or pharmacological target of the electron transport chain (ETC).
(2) We have developed technology to quantify ROS levels in vivo preserving the high resolution of in vitro measurements. This allows identifying which respiratory complex(es) produces ROS and how ROS are generated. Similarly, we have created models where the levels of superoxide and hydrogen peroxide are specifically modified, which allows identify the ROS responsible for specific physiological effects.
(3) The experimental increase in the reduction state of CoQ increases electron leak and ROS production at respiratory complex I (CI). We have dissected the mechanism by which ROS are increased showing that reverse electron transport (RET) is responsible for the increase in ROS. RET consists in the reverse transfer of electrons from ubiquinol to CI, where electrons are used to reduce back NAD+ to NADH generating a considerable amount of ROS during the process. Interestingly, the induction of RET-ROS (ROS produced via RET at CI) extends lifespan and protects mitochondrial function in old animals. This is the first time that RET-ROS is shown to extend lifespan in an animal model.
(4) RET-ROS can protect mitochondria against non-specific oxidative damage caused by depletion of Sod2 or blocking electron transfer within CI by mutations in pink1. Moreover, lifespan is strongly shortened in both mutants, and this phenotype is also rescued by inducing RET-ROS. The fact that RET-ROS rescues the phenotype caused by mutations Sod2 and pink1 does not support a major role of oxidative stress as the main cause for the pathology observed in these mutants. Similarly, we have shown that decreasing CI activity does not cause a Parkinson-like phenotype if RET-ROS is not interrupted. These results indicate that Parkinson's disease as ageing could be partially caused by the miscommunication between the mitochondrion and the rest of the cell.
(5) We have found an accumulation of free iron levels during ageing in Drosophila melanogaster. Interestingly, experimental increase of iron levels in young flies to the level observed in old flies do not accelerate ageing.
(6) We have found that suppression of ROS-RET signalling under stress conditions (including thermal and oxygen stresses) severely shortens survival of fruit flies. Interruption of ROS-RET signalling causes the accumulation of respiratory-deficient mitochondria as well as alterations in mitochondrial turnover and Target of rapamycin signalling.
(7) We have found that there is a specific transcriptional signature associated with stimulation or suppression of ROS-RET. Under non-stressed conditions stimulation of ROS-RET induces expression of pro-survival genes and pathways. Conversely, under stressed conditions suppression of ROS-RET causes the down-regulation of many pro-survival genes and pathways.
Exploitation Route This research project is increasing our knowledge about the role mitochondrial Reactive Oxygen Species (mtROS) play in vivo. We have shown that the place and the way mtROS are generated is instrumental to determining their physiological effects. For example, a controlled stimulation of ROS by complex I extends lifespan. We have described the first site-specific ROS signalling pathway that regulates lifespan in an animal. This will pave the way to describe other similar pathways regulating lifespan or other relevant physiological functions.
We have found that maintaining appropriate levels of ROS signalling is required to maintain cellular homeostasis and organismal survival under stress conditions. We foresee applications in our work to develop therapies targeting controlled production of ROS levels by mitochondrial complex I. These new drugs can target complex I or other complexes (e.g. III or IV) that stimulate reverse electron transport. Similarly, our research indicates that pharmaceutical companies must develop more specific antioxidants that prevent oxidative damage without interrupting redox signalling. In summary, we expect to see the translation of our research into new therapies to delay, prevent or reverse ageing and the onset of age-related diseases in humans.
Sectors Agriculture, Food and Drink,Chemicals,Healthcare,Leisure Activities, including Sports, Recreation and Tourism,Manufacturing, including Industrial Biotechology,Pharmaceuticals and Medical Biotechnology,Other

 
Description MRC Discover Medicine North (DiMeN) Doctoral Training Partnership
Amount £75,000 (GBP)
Organisation Medical Research Council (MRC) 
Sector Public
Country United Kingdom
Start 10/2017 
End 03/2021
 
Description Responsive Mode
Amount £328,974 (GBP)
Funding ID BB/R008167/1 
Organisation Biotechnology and Biological Sciences Research Council (BBSRC) 
Sector Public
Country United Kingdom
Start 05/2018 
End 04/2021
 
Description Senior Research Fellowship,
Amount £1,512,586 (GBP)
Funding ID 212241/Z/18/Z 
Organisation Wellcome Trust 
Sector Charity/Non Profit
Country United Kingdom
Start 05/2019 
End 04/2024
 
Description Sir Henry Wellcome Postdoctoral Fellowship
Amount £250,000 (GBP)
Organisation Wellcome Trust 
Sector Charity/Non Profit
Country United Kingdom
Start 10/2017 
End 09/2021
 
Title Humanised Ref(2)P 
Description Korolchuk and Sanz's laboratories used CRISPR/Cas9-Mediated Genome Editing to generate a humanised Ref(2)P (CG10360). Amino acids 91-116 of Drosophila (knock-in strain w1118) Ref(2)P were replaced with amino acids 100-118 of human p62/SQSTM1 peptide. 
Type Of Material Model of mechanisms or symptoms - non-mammalian in vivo 
Year Produced 2018 
Provided To Others? Yes  
Impact Replacement of endogenous Ref(2)P by humanised Ref(2)P in fruit flies increases protein turnover and stress resistance. This new model can be used to study autophagy and ageing as well as address evolutionary questions. 
URL https://www.ncbi.nlm.nih.gov/pmc/articles/PMC5772351/
 
Description Collaboration to study metabolic changes related with production of ROS via Reverse Electron Transport. 
Organisation University of Glasgow
Department Institute of Cancer Sciences
Country United Kingdom 
Sector Academic/University 
PI Contribution We have generated several fly models that produce different levels of ROS via Reverse Electron Transport (ROS-RET) and send fly brains to our collaborator Dr Oliver Maddocks (University of Glasgow) to study the metabolome.
Collaborator Contribution Our collaborators have studied the metabolome of flies producing different amount of ROS using a combination of targeted and untargeted metabolomics approaches.
Impact This collaborations has made possible understanding how cellular metabolism is re-wired to change the way respiratory complex I produces ROS from the forward to the reverse direction.
Start Year 2017
 
Description Collaboration to study the proteasome and redoxome in flies producing different amounts of mitochondrial Reactive Oxygen Species. 
Organisation National Center for Cardiovascular Research (CNIC)
Country Spain 
Sector Academic/University 
PI Contribution We have generated different fly models that produce different levels of ROS via Reverse Electron Transport (ROS-RET) and send fly brains to our collaborators Dr Enrique Calvo and Prof Jose Antonio Enriquez from the CNIC in Madrid (Spain).
Collaborator Contribution Our collaborators have produced the proteome and redoxome (i.e. set of proteins that have redox regulated cysteines). The proteome informs about changes in the levels of specific proteins in response to changes in ROS levels, while the redoxome allows detecting specific cysteine residues that are modified in the same experimental conditions.
Impact This collaboration is helping us understand which proteins are important in generating, transmitting and neutralizing ROS signals from the mitochondria.
Start Year 2017
 
Description Collaboration to study the transcriptome of long- and short-lived populations of Drosophila 
Organisation University of Leicester
Department MRC Toxicology Unit
Country United Kingdom 
Sector Public 
PI Contribution We have provided fly models to our collaborator Dr Miguel Martins from the MRC Toxicology Unit in Leicester. These models are characterized by producing higher levels of ROS and live longer than their controls or generate lower levels of ROS and live shorter than the controls.
Collaborator Contribution Our collaborators is helping us to analyse the data derived from RNA sequencing analysis. He is providing us with information about the genetic pathways altered in response to changes in mitochondrial ROS levels.
Impact Information about relevant genetic pathways altered in response to changes in ROS levels.
Start Year 2016
 
Description Co-organize and co-chair of Redox Signalling in Physiology, Ageing and Disease co-organized by the Biochemical Society and the British Society for Research on Ageing 
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 Organization of the scientific conference "Redox Signalling in Physiology, Ageing and Disease" supported by the Biochemical Society and the British Society for Research on Ageing with over 100 attendants.
Year(s) Of Engagement Activity 2019
URL https://www.eventsforce.net/biochemsoc/frontend/reg/thome.csp?pageID=20523&eventID=48&traceRedir=2
 
Description Teleconference in "Colegio Areteia" (School Areteia), Madrid, Spain 
Form Of Engagement Activity A talk or presentation
Part Of Official Scheme? No
Geographic Reach Regional
Primary Audience Schools
Results and Impact I gave different talks via skype to different classes of high school pupils (age range from 12-16). These talks aim to explain our research in ageing, the use of fruit flies in research and encourage young students to develop a scientific career. Also in collaboration with one of the teachers we are creating a web page together with the students that participated in the talks.The web page will explain the research of my group and how fruit flies are used for ageing and mitochondrial research.
Year(s) Of Engagement Activity 2016,2017,2019