New approaches to studying redox metabolism using time-resolved NAD(P)H fluorescence and anisotropy

Living cells require the constant input of energy to maintain their characteristic order. This is provided by the chemical reactions of metabolism. A vast number of these reactions are "redox" (reduction-oxidation) reactions, in which electrons are transferred from one molecule to another. During the breakdown of nutrient molecules to extract energy from food, bonds are broken and electrons are passed to a carrier molecule known as NADH. Removal of electrons is referred to as oxidation, and their addition is known as reduction. The metabolism of food therefore involves the oxidation of sugars, proteins and fats and the simultaneous reduction of NADH.

The energy passed to NADH during its reduction is converted into a useable form, the cell's "energy currency" adenosine triphosphate (ATP), inside the mitochondria. This is achieved by using the electrons carried by NADH to reduce oxygen to water. This is the fate of almost 100% of the oxygen consumed by the body, and the energy released is stored as ATP. Defects in this process cause the production of reactive oxygen species (ROS). These are highly damaging molecules, and so the cell possesses specific defence mechanisms against their deleterious effects. These defences are maintained by another electron carrier molecule known as NADPH.

The antioxidant systems supported by NADPH act to neutralise the harmful ROS produced during the energy generating processes regulated by NADH. These reactions are collectively known as redox metabolism. Imbalance between the two processes is a known factor in the development of a wide range of diseases, including cancer, diabetes and neurodegenerative disorders. In order to make progress in understanding how these diseases occur and testing potential treatments, it is crucial that biomedical researchers are provided with highly accurate tools to investigate redox metabolism in cells and tissues.

As a collection of scientists whose expertise includes the use of lasers to study the dynamic behaviour of molecules and the application of these methods to investigate metabolic processes in living tissues, we propose to develop the next generation of approaches to studying the role of redox metabolism in health and disease. We will exploit the intrinsic fluorescence of NADH and NADPH to construct our new experimental technique. While the two molecules emit light of the same colour, the two independent sets of enzymes that they bind to in order to perform their distinct roles will cause contrasting effects on other characteristics of their fluorescence.

We will first prepare solutions of NADH and NADPH bound to their respective enzymes to investigate the resulting properties of their fluorescence. The distinct binding sites to which these molecules attach may cause contrasting effects on the time taken for fluorescence to emerge following absorption of a laser pulse, the so-called fluorescence lifetime. Different forces acting on the molecules in the binding site will also cause differences in the freedom of their motion, which can be detected by measuring the rate of change of the polarisation of the light emitted with respect to the light absorbed, the so-called time-resolved fluorescence anisotropy.

Following characterisation of the differences in NADH and NADPH fluorescence when bound to their separate sets of enzymes, we will construct a microscope in which these properties can be detected inside living tissues. Inside the complex environment of the cell, the fluorescence signals would be expected to arise from a mixture of NADH and NADPH, both bound to their enzymes and free. We will develop approaches to extract information from these signals, allowing the function of the separate redox pathways that they are involved in to be investigated. As redox metabolism is being found to play a role in an ever growing range of processes, these new approaches will play a key role in enhancing our fundamental understanding of biology.

Redox processes play a key role in cell signalling, the fundamental process of ageing, and their incorrect regulation is implicated in the pathophysiology of numerous diseases. Biochemical measurements on cell extracts have provided the majority of findings on which the foundations of this field have been established. We aim to develop the next generation of tools to study redox processes in live tissues with spatial resolution, based on imaging the intrinsic fluorescence of the redox cofactors NADH and NADPH. NADH regulates energy-producing pathways while NADPH is primarily involved in the defence against reaction oxygen species. The emission spectra of the two cofactors are identical, ruling out the separate investigation of their distinct roles using conventional intensity based imaging. However, our previous work has suggested that the excited-state dynamics of these two cofactors differ within their separate binding sites. We will therefore perform a full characterisation of the enzyme-bound photophysics of NADH and NADPH, investigating the geometry and orientational freedom of the cofactors and their impacts on the fluorescence lifetime. This information will be used to refine the models we use to interpret measurements of the fluorescence decay of NAD(P)H in live tissues in terms of the underlying metabolism. Inside the environment of the cell, the fluorescence signal will result from a highly heterogeneous mix of NADH and NADPH, both free and bound to different sets of enzymes. In the study of such systems, time-resolved fluorescence intensity techniques are often unable to resolve the complex underlying dynamics and the measurement of additional fluorescence observables is necessary. To this end, we will develop a combined time-resolved fluorescence intensity and anisotropy imaging approach and apply it in the investigation of redox processes in a range of contexts including neuron-astrocyte metabolic coupling in the brain and redox metabolism in cancer.

Our proposal strongly reflects the 21st century research landscape in which boundaries must be broken down between traditional disciplines in order to investigate, understand and solve the most pressing issues in the health and wellbeing of the nation. Our interdisciplinary collaboration will develop and apply advanced time-resolved laser spectroscopy techniques to understand the role of redox processes in living tissues, such as in the fundamental process of ageing, and their contribution towards the development of diseases ranging from cancer to neurodegenerative disorders. In response to this, the societal and economic impact of this work will be sought in two key areas; the enhancement of public awareness of the key role interdisciplinary working plays in modern biological research, and knowledge transfer to academic and industrial developers of clinical diagnostic approaches. These are introduced below, and the means by which we will facilitate impact to potential beneficiaries are discussed in the attached Pathways to Impact document.

By using our research as a prime example of the interdisciplinary context in which modern biological research is taking place, engagement activities will ensure the public understanding of modern scientific approaches will be increased. Our work will demonstrate that the borders between disciplines introduced at school cease to exist at the cutting edge, so pupils considering a career in science need not abandon their interest in two of the three core disciplines to pursue the other. The early exposure to the overlaps between disciplines will inform the paths of these students through higher education, increasing the likelihood that the next generation of scientists will be produced with the wide knowledge and skills range for such research to take place. Achievement of this goal will have a positive impact on science in the United Kingdom and thus the economic prosperity of the nation.

Redox processes play a central role in the health and disease of biological tissues and the approaches we propose to develop will allow redox state to be interrogated in live tissues with molecular-level precision. As such, there is clear potential for these techniques to contribute to clinical diagnostics and screening. Instruments for measuring NAD(P)H fluorescence lifetimes both in situ in patients and in ex vivo biopsies have been created. However, their clinical application has been held back by lack of rigorous understanding of the links between the measured fluorescence signals and the underlying biochemistry. As this represents the central goal of our proposal, we will take an active stance in transferring the knowledge we gain to the university laboratories and companies attempting to develop these devices. While our primary academic goal is the development of these techniques for investigations into the fundamental role of redox processes in health and disease, we believe facilitating the transition of these approaches for clinical use represents a prime example of exploring impact outside our direct area of research to enhance the health and quality of life of the country.