Role of autophagy in the accumulation of defective mitochondria during ageing

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

Abstract

The progressive ageing of the UK population is one of the most serious problems our society will face during the 21st century. The increase in the number of people suffering from age-related diseases is a heavy burden on the NHS and threatens to collapse one of the best health care systems in the world.

The simplest and most cost effective solution to the "ageing problem" is to find ways to delay the rate of ageing, extending healthy lifespan, delaying the onset of age-related diseases and therefore extending the time an individual can remain fully independent. To delay ageing, we must fully understand the complex mechanisms that drive it and how they are connected.

Mitochondria are the powerhouses of the cell, generating the majority of ATP, the cellular currency required to keep cells going. As in any other factory, mitochondria generate harmful waste and require maintenance from time to time. Autophagy is the main way that the cell achieves this. It is responsible for removal and recycling of damaged components to keep the cell, including mitochondria, in optimal working condition. With age, the capacity of autophagy to do this declines. At the same time damaged mitochondria also accumulate, causing depletion of ATP, which leads to cell death and neurodegeneration and is associated with diseases such as Parkinson's or Alzheimer's disease.

We have found a new mechanism that connects the age associated reduction in autophagy efficiency and the accumulation of defective mitochondria. Importantly this mechanism can be targeted with drugs that are already in use in the clinic. When autophagy is interrupted, either genetically or during aging, respiratory complex I (CI) which mitochondria need to produce energy, stops working properly and starts to generate an abnormally high amount of harmful waste in the form of free radicals which can cause cell death and accelerate ageing. We have discovered that by-passing CI or restoring its function prevents cell death and extends lifespan.

We will develop a research program that will use a combination of in vitro and in vivo models to establish the connection between autophagy and mitochondria in ageing. Firstly, we will investigate how cell death is induced in autophagy-deficient cells, restoring CI function and rescuing cell death. Secondly, we will use the powerful genetics of the fruit fly to find out how loss of autophagy causes the accumulation of damaged mitochondria and measure it effect on the ageing process. Finally, we will boost autophagy and/or mitochondrial function to ask if by-passing CI in old individuals is enough to delay ageing. By the end of this project, we will have identified key mechanisms that drive ageing and can be targeted with therapies which are already clinically approved.

Technical Summary

Ageing is one of the most important biomedical challenges of the 21st century. Understanding the underlying mechanisms that drive ageing will provide us with the tools to solve the "ageing problem". Two hallmarks of ageing are diminished autopaghic capacity and accumulation of defective mitochondria.

Autophagy is instrumental for recycling damaged cellular components including mitochondria. Accordingly mutations in genes encoding the autophagy machinery cause severe neurodegeneration. Similarly, mitochondria are essential to produce cellular energy, and mutations in mitochondrial genes cause devastating diseases. Unfortunately, how autophagy dysfunction contributes to the accumulation of respiratory-deficient mitochondria in ageing remains to be established.

We have uncovered a new mechanism where genetic or age-related disruption of autophagy causes accumulation of dysfunctional mitochondria. These mitochondria generate less ATP and more Reactive Oxygen Species (ROS). Interestingly respiratory complex I (CI) is selectively affected in both autophagy-deficient cells and old individuals. CI is the largest respiratory enzyme and main generator of ROS, triggering cell death via apoptosis during dysfunction.

Our research program will determine how decreased autophagic capacity contributes to the accumulation of respiratory-deficient mitochondria and the role of CI in ageing. Firstly, we will investigate how CI triggers apoptosis. Secondly, we will determine how disrupted autophagy leads to the accumulation of CI-deficient mitochondria and how this affects ageing. Finally, we will implement genetic, pharmacological and dietary interventions to boost autophagy and restore CI function, extending healthy lifespan. Our project will provide us with specific targets that can be manipulated with clinically approved drugs, allowing a swift translation of our findings.

Planned Impact

Bioscience for health is one of the BBSRC strategic areas and within this area, extending population health span and increasing the independence of senior citizens is at the top of the list of priorities. The present consensus is that fundamental research directed at understanding the proximal causes of ageing is the most promising strategy to extend healthy lifespan. Our research program will operate under this paradigm determining the role autophagy and mitochondria play in ageing.

In the short term our research will have impact on ACADEMIC RESEARCHERS providing them with new knowledge about basic mechanisms of ageing. Our work will provide new tools and targets for drugs to CLINICIANS and HEALTH PROFESSIONALS working on ageing and degenerative diseases. A non-exclusive list includes: cancer, diabetes, immune diseases, Parkinson's disease and mitochondrial disease. In the long term, we aspire to develop treatments or interventions to prevent, delay or reverse ageing and extend healthy lifespan. Since both autophagy and mitochondria have been extensively studied, there are a number of clinically approved drugs (e.g. metformin, rapamycin or the bromodomain inhibitor I-BET 525762A).

Delaying ageing and age-related diseases will have an enormous IMPACT ON SOCIETY. One of the main problems the United Kingdom faces is the rising number of elderly people requiring more and more help to preserve their independence for longer. Alzheimer's disease, diabetes type II, sarcopenia, cancer or age-related cataracts are just a few examples of diseases and conditions that increase frailty and reduce the quality of life of the aged population. Finding a way to boost cellular energy levels in elderly citizens will contribute to this strategy by reducing frailty, extending their independence and dramatically reducing health care costs.

INDUSTRY will benefit from the possibility to perform drug screens in the in vivo and ex vivo models we will generate. Identifying novel or repurposing existing drugs that can prevent, delay or reverse the accumulation of dysfunctional mitochondria will open new markets, significantly increasing the revenue of the pharmaceutical industry. Ageing and age-related diseases will be a highly profitable market for pharmaceutical industry in the future. Our work will generate a list of drugs to be tested in higher model organisms or in humans. We will consider opportunities for commercialization in collaboration with the Newcastle University business development office who will evaluate the potential translational impact and industrial interaction of our research.

To boost the impact of our work, we will publish open access articles in high quality, broad readership journals and communicating our research via oral and poster presentations at scientific meetings before publication. We will take advantage of the websites of Newcastle University (including our own institute and laboratories), as well as social media (Twitter, Facebook) to actively promote our findings. The Newcastle University Press Office will be involved to ensure our findings are disseminated to the wider public as much as possible. Furthermore, we will generate opportunities throughout the project to disseminate and discuss our work with the general public, for whom the topic of healthy ageing is both accessible and interesting.

One of the most immediate impacts will be the CAREER DEVELOPMENT of the PDRA, RT and students who will learn and develop laboratory, analytical and writing skills, all of which are required for a successful career in science and to tackle age-related problems. Our cutting-edge research involving a number of latest TECHNOLOGIES will also train new SKILLED RESEARCHERS in UK.

Publications

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Related Projects

Project Reference Relationship Related To Start End Award Value
BB/R008167/1 01/05/2018 06/01/2020 £328,975
BB/R008167/2 Transfer BB/R008167/1 12/03/2020 30/06/2022 £175,197
 
Description Autophagy is the primary mechanism by which cells eliminate damaged components, including mitochondria. Elimination of damaged mitochondria is particularly crucial since they provide most of the cellular energy in the form of ATP. Reduced autophagy is a hallmark of ageing and age-related diseases. In parallel, ageing is characterized by the accumulation of defective mitochondria.

We have generated cellular models where autophagy is interrupted due to mutations in genes essential for the process such as atg5. Interestingly, autophagy-deficient cells survive without problems in a glucose-rich media where glycolysis is the leading way to generate ATP. However, the same cells die very fast when they are forced to use oxidative phosphorylation.

Our data indicate that autophagy deficiency triggers an NAD+ crisis in different cellular models. NAD+ is depleted due to its extensive use in repairing tasks (particularly DNA damaged by PARPs and sirtuins). Our data indicate that the "CI-defect" observed in autophagy-deficient cells is, in fact, an adaptation to diminished NAD+ levels and contributes to minimizing cell death.

Our results show that preventing NAD+ depletion (either boosting its synthesis or preventing its degradation) is the best strategy to reduce cell death in mammalian cellular models.

Interestingly, preventing the depletion of NAD(H) or boosting NAD+ synthesis is possible to rescue the lethal phenotype associated with autophagy deficiency in yeast, flies and human cells. Our discoveries open the door to new treatments for both ageing and age-related diseases.
Exploitation Route Our discoveries open the door to new treatments for both ageing and age-related diseases where autophagy is severely decreased. Boosting NAD+ levels is possible using compounds that are already available in the market. Our results support that the strategy works in yeast, Drosophila flies and human cells. The previous exciting results invite to test the same NAD+ boosters compounds in mouse models as a preliminary phase before planning clinical trials.
Sectors Healthcare,Pharmaceuticals and Medical Biotechnology

 
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 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
 
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 characterize the metabolome of cellular and fly models of autophagy dysfunction. 
Organisation University of Glasgow
Country United Kingdom 
Sector Academic/University 
PI Contribution We provide Dr Oliver Maddocks with different cellular models where autophagy is disrupted and cells are cultured in either glucose or galactose. In addition, we implement different treatments that improve survival of autophagy deficient cells. We aim to determine the metabolic changes behind such an improvement
Collaborator Contribution Combining targeted and untargeted metabolomics the laboratory of Dr Maddocks produces multiple metabolic measurements including the main metabolites belonging to glycolysis, Pentose Phosphate Pathway, Kreb's cycle, nucleotide metabolism, etc. that are altered in our cellular models of autophagy deficiency.
Impact This collaboration has made possible a better understanding of the metabolic changes caused by autophagy deficiency providing us with therapeutic targets to improve the deleterious phenotypes observed.
Start Year 2018
 
Description Collaboration to generate a neuronal model of autophagy deficiency. 
Organisation University of Birmingham
Country United Kingdom 
Sector Academic/University 
PI Contribution We provide Dr Sarkar with different models of autophagy deficiency cells and with expertise and information about mitochondrial function and techniques to evaluate mitochondrial activity, ROS production, etc.
Collaborator Contribution Dr Sarkar has generated human neurons differentiated from autophagy knockout human embryonic stem cells to test whether the phenotypes observed in fibroblast are conserved in this more relevant neurodegenerative disease model. In addition, we shall be able to test whether different therapeutic approaches to compensate autophagy deficiency also work in neurons.
Impact This collaboration provides us with an additional cellular model where to test whether the results observed in mouse fibroblasts also occur in another cellular type. Due to the fact, that many neurodegenerative diseases are associated with dysregulation of mechanism of quality control (such as autophagy), these neuronal models are highly relevant to validate new therapeutic targets.
Start Year 2018
 
Description Collaboration to measure superoxide levels ex vivo and in vivo. 
Organisation University of Cambridge
Country United Kingdom 
Sector Academic/University 
PI Contribution We use the superoxide detector provided by the laboratories of Profs Murphy and Hartley (MitoNeoD) to estimate superoxide levels in cells and fly tissues. We provide them with feedback about how the probe works in different cell types and fly tissues.
Collaborator Contribution Profs Harley and Murphy has provided us with MitoNeoD a modified version of MitoSox (a superoxide fluorescent probe) that fixes many of the problems associated with the latest e.g. lack of specificity, problems with DNA intercalation, etc.
Impact This collaboration allows a better estimation of the real levels of superoxide in our research models.
Start Year 2018
 
Description Collaboration to measure superoxide levels ex vivo and in vivo. 
Organisation University of Glasgow
Country United Kingdom 
Sector Academic/University 
PI Contribution We use the superoxide detector provided by the laboratories of Profs Murphy and Hartley (MitoNeoD) to estimate superoxide levels in cells and fly tissues. We provide them with feedback about how the probe works in different cell types and fly tissues.
Collaborator Contribution Profs Harley and Murphy has provided us with MitoNeoD a modified version of MitoSox (a superoxide fluorescent probe) that fixes many of the problems associated with the latest e.g. lack of specificity, problems with DNA intercalation, etc.
Impact This collaboration allows a better estimation of the real levels of superoxide in our research models.
Start Year 2018
 
Description Collaboration to measure the effect of disrupting autophagy in Saccharomyces cerevisae. 
Organisation Newcastle University
Country United Kingdom 
Sector Academic/University 
PI Contribution We provide knowledge to study the effects of autophagy disruption on Saccharomyces cerevisiae.
Collaborator Contribution The group lead by Dr Manolis Papamichos Chronakis has provided us with data showing the effect of disrupting autophagy in yeast and how the defect in growth can be partially rescued by providing the cells with NAD+ precursors.
Impact The experiments performed by Dr Papamichos Chronakis have shown that the response to disruption in autophagy is conserved from yeast to humans.
Start Year 2018
 
Description Collaboration to measure the impact of defects in autophagy on Caenorhabiditis elegans healthspan 
Organisation Durham University
Country United Kingdom 
Sector Academic/University 
PI Contribution We provide the team lead by Dr Weinkove with knowledge on how to generate models of autophagy deficiency in worms. Besides, we genotype the worms to confirm that specific genes were knocked down.
Collaborator Contribution Dr Weinkove provides us with measurements of healthspan of mutant and control worms using the automated system developed by his research group.
Impact We have obtained information about the effect of disrupting autophagy in worms (C. elegans)
Start Year 2019
 
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