The Flux Capacitor: How mitochondria modulate metabolic flux and gene expression

Mitochondria are often called the powerhouses of the cell as they produce nearly all the energy needed for living. Recent research shows that mitochondria do much more than generate energy: they integrate virtually all metabolic inputs and outputs of cells. These inputs and outputs depend on diet, temperature, growth, age and physical demands. If mitochondria cannot match metabolic supply to demand, they signal a stress state to the nucleus resulting in changes in the activity of genes. These changes might ameliorate the stress, or if that fails, tip the cell towards programmed death. Mitochondria can be seen as 'flux capacitors'.

The problem is that mitochondria are uniquely vulnerable to mechanical faults. This is because the vital proteins that carry out respiration are encoded by two different genomes that have a tendency to diverge - genes in the mitochondria mutate nearly 50 times faster than those in the nucleus, and are inherited from the mother only, whereas genes in the nucleus are recombined by sex every generation. These radical differences in inheritance can result in mismatches that affect the performance of mitochondria - the flux capacitor itself becomes faulty, which impacts on both the inputs and outputs of the cell, and its stress state.

Mutations in either the mitochondrial or nuclear genes encoding the proteins involved in respiration can cause catastrophic diseases, and more subtle genetic differences contribute to common conditions such as diabetes, cancer, neurodegeneration and ageing. But the extent to which mismatches between mitochondrial and nuclear genes affect health through the lifecourse is uncertain, as there are hundreds of mitochondria in every cell, and their performance can differ substantially. Animal models show that 'mitonuclear mismatches' really do affect health, for example causing male infertility and altered lifespan. Even when the health effects are too subtle to notice, mitonuclear mismatches can alter the activity of thousands of genes. Because these mismatches are produced every generation, they most likely have substantial health impacts. Until recently, though, this has been nearly impossible to verify.

Our programme of research will analyse how mitonuclear mismatches affect the inputs and outputs of mitochondria, and how these changes impact on gene activity and health. We will use a model organism, the fruitfly Drosophila, in which mitochondrial genes have been deliberately mismatched to the nuclear genome. These flies have known health outcomes such as male infertility, but how their faulty mitochondria cause these defects is unknown; treatments that might improve their health are unknown for the same reasons.

We will address these questions using cutting-edge experimental methods. Specifically, we will measure mitochondrial performance in real time to establish how mitonuclear mismatches alter the function of different tissues over the lifecourse of males and females, and how mitochondrial performance is altered by dietary treatments. We will generate global profiles of metabolite levels, allowing us to relate mitochondrial function to the 'metabolomic profile' of each tissue. Finally, we will use Next Generation Sequencing to measure changes in gene activity in each tissue, with each treatment, in males and females. We will use this information to build a set of mathematical models that map changes in mitochondrial function to shifts in metabolic flux, gene activity and health, allowing us to generalise our conclusions to be valuable for human and animal health.

Our pilot studies show that we can indeed measure real-time changes in mitochondrial performance linked with male infertility. Antioxidant treatments have remarkably different outcomes depending on mitonuclear mismatches, in one case improving male fertility yet causing high (90%) mortality in female flies. We therefore anticipate our findings will have important implications for lifelong health.

Mitochondria are central to metabolism but vulnerable to dysfunction, as respiratory proteins are encoded by two genomes (nuclear and mitochondrial) with radically different modes and tempi of evolution. Mitonuclear mismatches are produced each generation and can disrupt health, e.g. causing male infertility, altering lifespan and perturbing gene expression. But the incomplete penetrance of mitochondrial conditions means it has proved difficult to establish clear causality from alterations in mitochondrial function to changes in metabolic flux, shifts in gene expression and distinct phenotypes. Drosophila can give unparalleled insights into the consequences of mitonuclear mismatches, as these can be isolated from confounding factors that affect other models. We will therefore use a fly model to elucidate how mitonuclear incompatibilities affect (i) mitochondrial physiology (using high-resolution fluororespirometry on individual fly tissues), (ii) metabolomic profile, and (iii) gene expression (using Next Generation Sequencing) enabling us to (iv) construct predictive metabolic flux models for both males and females. We will use one coadapted control line and two experimental lines in which the mitochondrial genome is mismatched to the nuclear genome, combined with three treatments designed to interfere with signalling between the mitochondria and nucleus. Finally, we will assess how mitochondrial function, metabolomic profile, gene expression and phenotypes evolve over the lifecourse of flies. Our pilot data has already uncovered striking differences in response to the antioxidant NAC, determined by mitonuclear incompatibilities; in one line male fertility improved but 90% of females died; NAC did not affect mortality in other lines. We therefore anticipate that our work will generate fundamental biological knowledge on how mitonuclear variations cause differences in physiology, wellbeing, drug interactions and lifelong susceptibility to disease between individuals.

Who will benefit from this research?

Our considerations have highlighted seven particular groups of end users that we wish to target through our pathway to impact activities:

1) Investigators and scholars of the evolutionary history of eukaryotes (Academic/Public Sector Researchers e.g. Evolutionary Biologists)
2) Researchers and other stakeholders of eukaryote based biotechnological applications (Academic/Public Sector Researchers, Organisations e.g. Bioprocessing Research Industry Club (BRIC), and Private Sector/Industry)
3) Medical researchers of mitochondrial, metabolic, and ageing related diseases (Academic/Public Sector Researchers, Medical Practitioners)
4) Health policy makers and government bodies interested in increasing general population quality of life through healthy ageing. (Public Sector Health Researchers e.g. NIHR related, Public Sector Health Policy Makers e.g. NIHR, HRA and Department of Health)
5) Regulatory policy makers and regulatory body for treatments impacting on mitochondrial function and metabolism. (Public Sector Regulator e.g. EMA, MHRA)
6) Researchers, producers and distributors of nutritional supplements that may impact health via mitochondrial interventions. (Private Sector Stakeholders e.g. Pharma/Nutritional Supplement developers/producers)
7) Members of the public interested in mitochondria and healthy ageing guidelines. (General Public)

How will they benefit from this research?

Researchers interested in mitonuclear evolution and metabolism will gain insights from our data. Not only will we produce empirical data that with give vital insights into mitochondrial metabolism, but we will merge all the empirical data to produce a flux model. What we will deliver from this grant will be of interest to a wide range of scientists; from evolutionary ecologists to functional biologists.

Biomedical scientists, policy makers and government bodies will be interested in our findings because of their relevance to human complex diseases (the accumulation and impact of mitochondrial mutations), the disruption of adaptive mitonuclear combinations by human migration (the scope for the coevolution of beneficial mitonuclear combinations) and the limits of mitonuclear compatibility. Biomedical researchers and policy makers will rely on such data in order to develop rigorous, but sensitive, management of medical interventions, including mitochondrial replacement therapy and three-parent IVF, and to establish appropriate policies that regulate these applications.

By addressing the role of mitochondrial function in supporting healthy ageing this work could influence not only researchers but also policy makers and clinical practitioners, with consequent impact upon quality of life for the wider public. Moreover, our work will touch upon the effects of mitonuclear genomic incompatibility in determining health outcomes from pharmacological or other medical interventions. This could influence not only those involved in the research, development, and production of such interventions but also those tasked with setting and consequently those adhering to policy on medical and pharmacological regulation.

Finally, our research will be of broad interest to the wider community and school pupils. They have a natural fascination in mitochondrial disease and mitochondrial replacement therapy as a cure. The project findings will be made readily available to the general public and the project team will engage directly with schools, to make sure that the project findings are placed into a broad context of evolution and the life sciences.