Using exomarkers to assess mitochondrial reactive species in vivo

Background: The ability to measure the concentrations of small damaging and signalling molecules such as reactive oxygen species (ROS) in vivo is essential to understanding their biological roles. While a range of methods can be applied to in vitro systems, measuring the levels and relative changes in reactive species in vivo is challenging. Scope of review: One approach towards achieving this goal is the use of exomarkers. In this, exogenous probe compounds are administered to the intact organism and are then transformed by the reactive molecules in vivo to produce a diagnostic exomarker. The exomarker and the precursor probe can be analysed ex vivo to infer the identity and amounts of the reactive species present in vivo. This is akin to the measurement of biomarkers produced by the interaction of reactive species with endogenous biomolecules. Major conclusions and general significance: Our laboratories have developed mitochondria-targeted probes that generate exomarkers that can be analysed ex vivo by mass spectrometry to assess levels of reactive species within mitochondria in vivo. We have used one of these compounds, MitoB, to infer the levels of mitochondrial hydrogen peroxide within flies and mice. Here we describe the development of MitoB and expand on this example to discuss how better probes and exomarkers can be developed. This article is part of a Special Issue entitled Current methods to study reactive oxygen species - pros and cons and biophysics of membrane proteins. Guest Editor: Christine Winterbourn. Abbreviations: EPR, electron paramagnetic resonance; GFP, green fluorescent protein; 4-HNE, 4-hydroxynonenal; MitoB, 3-(dihydroxyboronyl)benzyltriphenylphosphonium bromide; MitoP, (3-hydroxybenzyl)triphenylphosphonium bromide; ROS, reactive oxygen species; SOD, superoxide dismutase; TPMP, methyltriphenylphosphonium; TPP, triphenylphosphonium catio

Measurement of H₂O₂ within living drosophila during aging using a ratiometric mass spectrometry probe targeted to the mitochondrial matrix

Hydrogen peroxide (H<sub>2</sub>O<sub>2</sub>) is central to mitochondrial oxidative damage and redox signaling, but its roles are poorly understood due to the difficulty of measuring mitochondrial H<sub>2</sub>O<sub>2</sub> in vivo. Here we report a ratiometric mass spectrometry probe approach to assess mitochondrial matrix H<sub>2</sub>O<sub>2</sub> levels in vivo. The probe, MitoB, comprises a triphenylphosphonium (TPP) cation driving its accumulation within mitochondria, conjugated to an arylboronic acid that reacts with H<sub>2</sub>O<sub>2</sub> to form a phenol, MitoP. Quantifying the MitoP/MitoB ratio by liquid chromatography-tandem mass spectrometry enabled measurement of a weighted average of mitochondrial H<sub>2</sub>O<sub>2</sub> that predominantly reports on thoracic muscle mitochondria within living flies. There was an increase in mitochondrial H<sub>2</sub>O<sub>2</sub> with age in flies, which was not coordinately altered by interventions that modulated life span. Our findings provide approaches to investigate mitochondrial ROS in vivo and suggest that while an increase in overall mitochondrial H<sub>2</sub>O<sub>2</sub> correlates with aging, it may not be causative

Enhancing autophagy by redox regulation extends lifespan in <i>Drosophila</i>

Redox signalling is an important modulator of diverse biological pathways and processes, and operates through specific post-translational modification of redox-sensitive thiols on cysteine residues 1–4. Critically, redox signalling is distinct from irreversible oxidative damage and functions as a reversible ‘redox switch’ to regulate target proteins. H2O2 acts as the major effector of redox signalling, both directly and through intracellular thiol redox relays 5,6. Dysregulation of redox homeostasis has long been implicated in the pathophysiology of many age-related diseases, as well as in the ageing process itself, however the underlying mechanisms remain largely unclear 7,8. To study redox signalling by H2O2in vivo and explore its involvement in metabolic health and longevity, we used the fruit fly Drosophila as a model organism, with its tractable lifespan and strong evolutionary conservation with mammals 9. Here we report that inducing an endogenous redox-shift, by manipulating levels of the H2O2-degrading enzyme catalase, improves health and robustly extends lifespan in flies, independently of oxidative stress resistance and dietary restriction. We find that the catalase redox-shifted flies are acutely sensitive to starvation stress, which relies on autophagy as a vital survival mechanism. Importantly, we show that autophagy is essential for the lifespan extension of the catalase flies. Furthermore, using redox-inactive knock-in mutants of Atg4a, a major effector of autophagy, we show that the lifespan extension in response to catalase requires a key redox-regulatory cysteine residue, Cys102 in Atg4a. These findings demonstrate that redox regulation of autophagy can extend lifespan, confirming the importance of redox signalling in ageing and as a potential pro-longevity target.</jats:p

Oxidative stress and life histories: unresolved issues and current needs.

Life-history theory concerns the trade-offs that mold the patterns of investment by animals between reproduction, growth, and survival. It is widely recognized that physiology plays a role in the mediation of life-history trade-offs, but the details remain obscure. As life-history theory concerns aspects of investment in the soma that influence survival, understanding the physiological basis of life histories is related, but not identical, to understanding the process of aging. One idea from the field of aging that has gained considerable traction in the area of life histories is that life-history trade-offs may be mediated by free radical production and oxidative stress. We outline here developments in this field and summarize a number of important unresolved issues that may guide future research efforts. The issues are as follows. First, different tissues and macromolecular targets of oxidative stress respond differently during reproduction. The functional significance of these changes, however, remains uncertain. Consequently there is a need for studies that link oxidative stress measurements to functional outcomes, such as survival. Second, measurements of oxidative stress are often highly invasive or terminal. Terminal studies of oxidative stress in wild animals, where detailed life-history information is available, cannot generally be performed without compromising the aims of the studies that generated the life-history data. There is a need therefore for novel non-invasive measurements of multi-tissue oxidative stress. Third, laboratory studies provide unrivaled opportunities for experimental manipulation but may fail to expose the physiology underpinning life-history effects, because of the benign laboratory environment. Fourth, the idea that oxidative stress might underlie life-history trade-offs does not make specific enough predictions that are amenable to testing. Moreover, there is a paucity of good alternative theoretical models on which contrasting predictions might be based. Fifth, there is an enormous diversity of life-history variation to test the idea that oxidative stress may be a key mediator. So far we have only scratched the surface. Broadening the scope may reveal new strategies linked to the processes of oxidative damage and repair. Finally, understanding the trade-offs in life histories and understanding the process of aging are related but not identical questions. Scientists inhabiting these two spheres of activity seldom collide, yet they have much to learn from each other

Fine-tuning autophagy maximises lifespan and is associated with changes in mitochondrial gene expression in Drosophila

Increased cellular degradation by autophagy is a feature of many interventions that delay ageing. We report here that increased autophagy is necessary for reduced insulin-like signalling (IIS) to extend lifespan in Drosophila and is sufficient on its own to increase lifespan. We first established that the well-characterised lifespan extension associated with deletion of the insulin receptor substrate chico was completely abrogated by downregulation of the essential autophagy gene Atg5. We next directly induced autophagy by over-expressing the major autophagy kinase Atg1 and found that a mild increase in autophagy extended lifespan. Interestingly, strong Atg1 up-regulation was detrimental to lifespan. Transcriptomic and metabolomic approaches identified specific signatures mediated by varying levels of autophagy in flies. Transcriptional upregulation of mitochondrial-related genes was the signature most specifically associated with mild Atg1 upregulation and extended lifespan, whereas short-lived flies, possessing strong Atg1 overexpression, showed reduced mitochondrial metabolism and up-regulated immune system pathways. Increased proteasomal activity and reduced triacylglycerol levels were features shared by both moderate and high Atg1 overexpression conditions. These contrasting effects of autophagy on ageing and differential metabolic profiles highlight the importance of fine-tuning autophagy levels to achieve optimal healthspan and disease prevention

Fine-tuning autophagy maximises lifespan and is associated with changes in mitochondrial gene expression in Drosophila

Increased cellular degradation by autophagy is a feature of many interventions that delay ageing. We report here that increased autophagy is necessary for reduced insulin-like signalling (IIS) to extend lifespan in Drosophila and is sufficient on its own to increase lifespan. We first established that the well-characterised lifespan extension associated with deletion of the insulin receptor substrate chico was completely abrogated by downregulation of the essential autophagy gene Atg5. We next directly induced autophagy by over-expressing the major autophagy kinase Atg1 and found that a mild increase in autophagy extended lifespan. Interestingly, strong Atg1 up-regulation was detrimental to lifespan. Transcriptomic and metabolomic approaches identified specific signatures mediated by varying levels of autophagy in flies. Transcriptional upregulation of mitochondrial-related genes was the signature most specifically associated with mild Atg1 upregulation and extended lifespan, whereas short-lived flies, possessing strong Atg1 overexpression, showed reduced mitochondrial metabolism and up-regulated immune system pathways. Increased proteasomal activity and reduced triacylglycerol levels were features shared by both moderate and high Atg1 overexpression conditions. These contrasting effects of autophagy on ageing and differential metabolic profiles highlight the importance of fine-tuning autophagy levels to achieve optimal healthspan and disease prevention

Host-Microbe-Drug-Nutrient Screen Identifies Bacterial Effectors of Metformin Therapy.

Metformin is the first-line therapy for treating type 2 diabetes and a promising anti-aging drug. We set out to address the fundamental question of how gut microbes and nutrition, key regulators of host physiology, affect the effects of metformin. Combining two tractable genetic models, the bacterium E. coli and the nematode C. elegans, we developed a high-throughput four-way screen to define the underlying host-microbe-drug-nutrient interactions. We show that microbes integrate cues from metformin and the diet through the phosphotransferase signaling pathway that converges on the transcriptional regulator Crp. A detailed experimental characterization of metformin effects downstream of Crp in combination with metabolic modeling of the microbiota in metformin-treated type 2 diabetic patients predicts the production of microbial agmatine, a regulator of metformin effects on host lipid metabolism and lifespan. Our high-throughput screening platform paves the way for identifying exploitable drug-nutrient-microbiome interactions to improve host health and longevity through targeted microbiome therapies. VIDEO ABSTRACT

Mitochondrial pharmacology.

Mitochondria are being recognized as key factors in many unexpected areas of biomedical science. In addition to their well-known roles in oxidative phosphorylation and metabolism, it is now clear that mitochondria are also central to cell death, neoplasia, cell differentiation, the innate immune system, oxygen and hypoxia sensing, and calcium metabolism. Disruption to these processes contributes to a range of human pathologies, making mitochondria a potentially important, but currently seemingly neglected, therapeutic target. Mitochondrial dysfunction is often associated with oxidative damage, calcium dyshomeostasis, defective ATP synthesis, or induction of the permeability transition pore. Consequently, therapies designed to prevent these types of damage are beneficial and can be used to treat many diverse and apparently unrelated indications. Here we outline the biological properties that make mitochondria important determinants of health and disease, and describe the pharmacological strategies being developed to address mitochondrial dysfunction

Oxidative stress and life histories: unresolved issues and current needs.

Life-history theory concerns the trade-offs that mold the patterns of investment by animals between reproduction, growth, and survival. It is widely recognized that physiology plays a role in the mediation of life-history trade-offs, but the details remain obscure. As life-history theory concerns aspects of investment in the soma that influence survival, understanding the physiological basis of life histories is related, but not identical, to understanding the process of aging. One idea from the field of aging that has gained considerable traction in the area of life histories is that life-history trade-offs may be mediated by free radical production and oxidative stress. We outline here developments in this field and summarize a number of important unresolved issues that may guide future research efforts. The issues are as follows. First, different tissues and macromolecular targets of oxidative stress respond differently during reproduction. The functional significance of these changes, however, remains uncertain. Consequently there is a need for studies that link oxidative stress measurements to functional outcomes, such as survival. Second, measurements of oxidative stress are often highly invasive or terminal. Terminal studies of oxidative stress in wild animals, where detailed life-history information is available, cannot generally be performed without compromising the aims of the studies that generated the life-history data. There is a need therefore for novel non-invasive measurements of multi-tissue oxidative stress. Third, laboratory studies provide unrivaled opportunities for experimental manipulation but may fail to expose the physiology underpinning life-history effects, because of the benign laboratory environment. Fourth, the idea that oxidative stress might underlie life-history trade-offs does not make specific enough predictions that are amenable to testing. Moreover, there is a paucity of good alternative theoretical models on which contrasting predictions might be based. Fifth, there is an enormous diversity of life-history variation to test the idea that oxidative stress may be a key mediator. So far we have only scratched the surface. Broadening the scope may reveal new strategies linked to the processes of oxidative damage and repair. Finally, understanding the trade-offs in life histories and understanding the process of aging are related but not identical questions. Scientists inhabiting these two spheres of activity seldom collide, yet they have much to learn from each other