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Despite longstanding scientific interest in the centrality of mitochondria to biological aging, directly controlling mitochondrial function to test causality has eluded researchers. We show that specifically boosting mitochondrial membrane potential through a light-activated proton pump reversed age-associated phenotypes and extended C. elegans lifespan. We show that harnessing the energy of light to experimentally increase mitochondrial membrane potential during adulthood alone is sufficient to slow the rate of aging.

Organisms rely on chemical cues in their environment to indicate the presence or absence of food, reproductive partners, predators, or other harmful stimuli. In the nematode Caenorhabditis elegans, the bilaterally symmetric pair of ASH sensory neurons serves as the primary nociceptors. ASH activation by aversive stimuli leads to backward locomotion and stimulus avoidance. We previously reported a role for guanylyl cyclases in dampening nociceptive sensitivity that requires an innexin-based gap junction network to pass cGMP between neurons. Here, we report that animals lacking function of the gap junction component INX-20 are hypersensitive in their behavioral response to both soluble and volatile chemical stimuli that signal through G protein-coupled receptor pathways in ASH. We find that expressing inx-20 in the ADL and AFD sensory neurons is sufficient to dampen ASH sensitivity, which is supported by new expression analysis of endogenous INX-20 tagged with mCherry via the CRISPR-Cas9 system. Although ADL does not form gap junctions directly with ASH, it does so via gap junctions with the interneuron RMG and the sensory neuron ASK. Ablating either ADL or RMG and ASK also resulted in nociceptive hypersensitivity, suggesting an important role for RMG/ASK downstream of ADL in the ASH modulatory circuit. This work adds to our growing understanding of the repertoire of ways by which ASH activity is regulated via its connectivity to other neurons and identifies a previously unknown role for ADL and RMG in the modulation of aversive behavior.

Hydrogen peroxide (H2O2) functions as a second messenger to signal metabolic distress through highly compartmentalized production in mitochondria. The dynamics of ROS generation and diffusion between mitochondrial compartments and into the cytosol govern oxidative stress responses and pathology, though our understanding of these processes remains limited. Here, we couple the H2O2biosensor, HyPer7, with optogenetic stimulation of the ROS-generating protein KillerRed targeted into multiple mitochondrial microdomains. Single mitochondrial photogeneration of H2O2demonstrates the spatiotemporal dynamics of ROS diffusion and transient hyperfusion of mitochondria due to ROS. Measurement of microdomain-specific H2O2diffusion kinetics reveals directionally selective diffusion through mitochondrial microdomains. All-optical generation and detection of physiologically-relevant concentrations of H2O2between mitochondrial compartments provide a map of mitochondrial H2O2diffusion dynamicsin situ. These kinetic details of spatiotemporal ROS dynamics and inter-mitochondrial spreading forms a framework to understand the role of ROS in health and disease.

Developing new technologies to study metabolism is increasingly important as metabolic disease prevalence increases. Mitochondria control cellular metabolism and dynamic changes in mitochondrial function are associated with metabolic abnormalities in cardiovascular disease, cancer, and obesity. However, a lack of precise and reversible methods to control mitochondrial function has prevented moving from association to causation. Recent advances in optogenetics have addressed this challenge, and mitochondrial function can now be precisely controlled in vivo using light. A class of genetically-encoded, light-activated membrane channels and pumps has addressed mechanistic questions that promise to provide new insights into how cellular metabolism downstream of mitochondrial function contributes to disease. Here, we highlight emerging reagents – mitochondria-targeted light-activated cation channels or proton pumps – to decrease or increase mitochondrial activity upon light exposure, a technique we refer to as mitochondrial light switches, or mt(SWITCH). The mt(SWITCH) technique is broadly applicable, as energy availability and metabolic signaling are conserved aspects of cellular function and health. Here, we outline the use of these tools in diverse cellular models of disease. We review the molecular details of each optogenetic tool, summarize the results obtained with each, and outline best practices for using optogenetic approaches to control mitochondrial function and downstream metabolism.

Mitochondria are essential for neuronal survival and mitochondrial dysfunction is a hallmark of neurodegeneration. The loss in mitochondrial energy production, oxidative stress, and changes in calcium handling are associated with neurodegenerative diseases; however, different sites and types of mitochondrial dysfunction are linked to distinct neuropathologies. Understanding the causal or correlative relationship between changes in mitochondria and neuropathology will lead to new therapeutic strategies. Here, we summarize the evidence of site-specific mitochondrial dysfunction and mitochondrial-related clinical trials for neurodegenerative diseases. We further discuss potential therapeutic approaches, such as mitochondrial transplantation, restoration of mitochondrial function, and pharmacological alleviation of mitochondrial dysfunction.

Aims: How mitochondrial reactive oxygen species (ROS) impact physiological function may depend on the quantity of ROS generated or removed, and the subcellular microdomain in which this oc...

C. elegans react to metabolic distress caused by mismatches in oxygen and energy status via distinct behavioral responses. At the molecular level, these responses are coordinated by under-characterized, redox-sensitive processes, thought to initiate in mitochondria. Complex I of the electron transport chain is a major site of reactive oxygen species (ROS) production and is canonically associated with oxidative damage following hypoxic exposure. Here, we use a combination of optogenetics and CRISPR/Cas9-mediated genome editing to exert spatiotemporal control over ROS production. We demonstrate a photo-locomotory remodeling of avoidance behavior by local ROS production due to the reversible oxidation of a single thiol on the complex I subunit NDUF-2.1. Reversible thiol oxidation at this site is necessary and sufficient for the behavioral response to hypoxia, does not respond to ROS produced at more distal sites, and protects against lethal hypoxic exposure. Molecular modeling suggests that oxidation at this thiol residue alters the ability for NDUF-2.1 to coordinate electron transfer to coenzyme Q by destabilizing the Q-binding pocket, causing decreased complex I activity. Overall, site-specific ROS production regulates behavioral responses and these findings provide a mechanistic target to suppress the detrimental effects of hypoxia.

Organisms adapt to their environment through coordinated changes in mitochondrial function and metabolism. The mitochondrial protonmotive force (PMF) is an electrochemical gradient that powers ATP synthesis and adjusts metabolism to energetic demands via cellular signaling. It is unknown how or where transient PMF changes are sensed and signaled due to the lack of precise spatiotemporal control in vivo. We addressed this by expressing a light‐activated proton pump in mitochondria to spatiotemporally “turn off” mitochondrial function through PMF dissipation in tissues with light. We applied our construct—mitochondria‐OFF (mtOFF)—to understand how metabolic status impacts hypoxia resistance, a response that relies on mitochondrial function. Activation of mtOFF induced starvation‐like behavior mediated by AMP‐activated protein kinase (AMPK). We found prophylactic mtOFF activation increased survival following hypoxia, and that protection relied on neuronal AMPK. Our study links spatiotemporal control of mitochondrial PMF to cellular metabolic changes that mediate behavior and stress resistance.

Organisms adapt to their environment through coordinated changes in mitochondrial function and metabolism. The mitochondrial protonmotive force (PMF) is an electrochemical gradient that powers ATP synthesis and adjusts metabolism to energetic demands via cellular signaling. It is unknown how or where transient PMF changes are sensed and signaled due to lack of precise spatiotemporal control in vivo. We addressed this by expressing a light-activated proton pump in mitochondria to spatiotemporally “turn off” mitochondrial function through PMF dissipation in tissues with light. We applied our construct – mitochondria-OFF (mtOFF) – to understand how metabolic status impacts hypoxia resistance, a response that relies on mitochondrial function. mtOFF activation induced starvation-like behavior mediated by AMP-activated protein kinase (AMPK). We found prophylactic mtOFF activation increased survival following hypoxia, and that protection relied on neuronal AMPK. Our study links spatiotemporal control of mitochondrial PMF to cellular metabolic changes that mediate behavior and stress resistance.

Oxidants play an important role in the cell and are involved in many redox processes. Oxidant concentrations are maintained through coordinated production and removal systems. The dysregulation of oxidant homeostasis is a hallmark of many disease pathologies. The local oxidant microdomain is crucial for the initiation of many redox signaling events; however, methods to control oxidant product are limited. Some fluorescent proteins, including GFP, TagRFP, KillerRed, miniSOG, and their derivatives, generate oxidants in response to light. These genetically-encoded photosensitizers produce singlet oxygen and superoxide upon illumination and offer spatial and temporal control over oxidant production. In this review, we will examine the photosensitization properties of fluorescent proteins and their application to redox biology. Emerging concepts of selective oxidant species production via photosensitization and the impact of light on biological systems are discussed.

The cell maintains a balance between the production and removal of reactive oxygen species (ROS). Changes in ROS levels can impact many cellular functions, and dysregulation contributes to pathologies. How a specific cellular environment or microdomain influences the ROS-generating systems and biological impact of ROS remains an active area of research. This Forum highlights the complexity of ROS microdomains and their contributions to health and disease. Novel technologies to measure or generate ROS in defined regions are important developments in the spatial control of ROS. Using these advances, the articles herein demonstrate how site-specific redox environments influence cellular function and pathology. Antioxid. Redox Signal. 31, 591–593.

Genetically-encoded photosensitizers produce reactive oxygen species (ROS) in response to light. Transgenic expression of fusion proteins can target the photosensitizers to specific cell regions and permit the spatial and temporal control of ROS production. These ROS-generating proteins (RGPs) are widely used for cell ablation, mutagenesis and chromophore-assisted light inactivation of target proteins. However, the species produced by RGPs are unclear due to indirect measures with confounding interpretations. Recently, the RGP mini “Singlet Oxygen Generator” (miniSOG) was engineered from Arabidopsis thaliana phototropin 2. While miniSOG produces singlet oxygen ((1)O(2)), the contribution of superoxide (O(2)(•−)) to miniSOG-generated ROS remains unclear. We measured the light-dependent O(2)(•−) production of purified miniSOG using HPLC separation of dihydroethidium (DHE) oxidation products. We demonstrate that DHE is insensitive to (1)O(2) and establish that DHE is a suitable indicator to measure O(2)(•−) production in a system that produces both (1)O(2) and O(2)(•−). We report that miniSOG produces both (1)O(2) and O(2)(•−), as can its free chromophore, flavin mononucleotide. miniSOG produced O(2)(•−) at a rate of ~4.0 μmol O(2)(•−)/min/μmol photosensitizer for an excitation fluence rate of 5.9 mW/mm(2) at 470 ± 20 nm, and the rate remained consistent across fluences (light doses). Overall, the contribution of O(2)(•−) to miniSOG phenotypes should be considered.

Mitochondrial respiration generates an electrochemical proton gradient across the mitochondrial inner membrane called protonmotive force (PMF) to drive diverse functions and synthesize ATP. Current techniques to manipulate the PMF are limited to its dissipation; yet, there is no precise and reversible method to increase the PMF. To address this issue, we aimed to use an optogenetic approach and engineered a mitochondria‐targeted light‐activated proton pump that we name mitochondria‐ON (mtON) to selectively increase the PMF in Caenorhabditis elegans. Here we show that mtON photoactivation increases the PMF in a dose‐dependent manner, supports ATP synthesis, increases resistance to mitochondrial toxins, and modulates energy‐sensing behavior. Moreover, transient mtON activation during hypoxic preconditioning prevents the well‐characterized adaptive response of hypoxia resistance. Our results show that optogenetic manipulation of the PMF is a powerful tool to modulate metabolism and cell signaling.

Fluorescent proteins can generate reactive oxygen species (ROS) upon absorption of photons via type I and II photosensitization mechanisms. The red fluorescent proteins KillerRed and SuperNova are phototoxic proteins engineered to generate ROS and are used in a variety of biological applications. However, their relative quantum yields and rates of ROS production are unclear, which has limited the interpretation of their effects when used in biological systems. We cloned and purified KillerRed, SuperNova, and mCherry - a related red fluorescent protein not typically considered a photosensitizer - and measured the superoxide (O2•-) and singlet oxygen (1O2) quantum yields with irradiation at 561 nm. The formation of the O2•--specific product 2-hydroxyethidium (2-OHE+) was quantified via HPLC separation with fluorescence detection. Relative to a reference photosensitizer, Rose Bengal, the O2•- quantum yield (ΦO2•-) of SuperNova was determined to be 0.00150, KillerRed was 0.00097, and mCherry 0.00120. At an excitation fluence of 916.5 J/cm2 and matched absorption at 561 nm, SuperNova, KillerRed and mCherry made 3.81, 2.38 and 1.65 μM O2•-/min, respectively. Using the probe Singlet Oxygen Sensor Green (SOSG), we ascertained the 1O2 quantum yield (Φ1O2) for SuperNova to be 0.0220, KillerRed 0.0076, and mCherry 0.0057. These photosensitization characteristics of SuperNova, KillerRed and mCherry improve our understanding of fluorescent proteins and are pertinent for refining their use as tools to advance our knowledge of redox biology.GRAPHICAL ABSTRACT

Mitochondrial respiration generates an electrochemical proton gradient across the mitochondrial inner membrane called the protonmotive force (PMF) to drive diverse functions and make ATP. Current techniques to manipulate the PMF are limited to its dissipation; there is no precise, reversible method to increase the PMF. To address this issue, we used an optogenetic approach and engineered a mitochondria-targeted light-activated proton pumping protein we called mitochondria-ON (mtON) to selectively increase the PMF. Here, mtON increased the PMF light dose-dependently, supported ATP synthesis, increased resistance to mitochondrial toxins, and modulated energy-sensing behavior in Caenorhabditis elegans. Moreover, transient mtON activation during hypoxia prevented the well-characterized adaptive response of hypoxic preconditioning. Our novel optogenetic approach demonstrated that a decreased PMF is both necessary and sufficient for hypoxia-stimulated stress resistance. Our results show that optogenetic manipulation of the PMF is a powerful tool to modulate metabolic and cell signaling outcomes.

Mitochondrial reactive oxygen species (ROS) can be either detrimental or beneficial depending on the amount, duration, and location of their production. Mitochondrial complex I is a component of the electron transport chain and transfers electrons from NADH to ubiquinone. Complex I is also a source of ROS production. Under certain thermodynamic conditions, electron transfer can reverse direction and reduce oxygen at complex I to generate ROS. Conditions that favor this reverse electron transport (RET) include highly reduced ubiquinone pools, high mitochondrial membrane potential, and accumulated metabolic substrates. Historically, complex I RET was associated with pathological conditions, causing oxidative stress. However, recent evidence suggests that ROS generation by complex I RET contributes to signaling events in cells and organisms. Collectively, these studies demonstrate that the impact of complex I RET, either beneficial or detrimental, can be determined by the timing and quantity of ROS production. In this article we review the role of site-specific ROS production at complex I in the contexts of pathology and physiologic signaling.

Adhesion G protein–coupled receptors (aGPCRs) have emerging roles in development and tissue maintenance and is the most prevalent GPCR subclass mutated in human cancers, but to date, no drugs have been developed to target them in any disease. aGPCR extracellular domains contain a conserved subdomain that mediates self-cleavage proximal to the start of the 7-transmembrane domain (7TM). The two receptor protomers, extracellular domain and amino terminal fragment (NTF), and the 7TM or C-terminal fragment remain noncovalently bound at the plasma membrane in a low-activity state. We recently demonstrated that NTF dissociation liberates the 7TM N-terminal stalk, which acts as a tethered-peptide agonist permitting receptor-dependent heterotrimeric G protein activation. In many cases, natural aGPCR ligands are extracellular matrix proteins that dissociate the NTF to reveal the tethered agonist. Given the perceived difficulty in modifying extracellular matrix proteins to create aGPCR probes, we developed a serum response element (SRE)-luciferase–based screening approach to identify GPR56/ADGRG1 small-molecule inhibitors. A 2000-compound library comprising known drugs and natural products was screened for GPR56-dependent SRE activation inhibitors that did not inhibit constitutively active Gα13-dependent SRE activation. Dihydromunduletone (DHM), a rotenoid derivative, was validated using cell-free aGPCR/heterotrimeric G protein guanosine 5′-3-O-(thio)triphosphate binding reconstitution assays. DHM inhibited GPR56 and GPR114/ADGRG5, which have similar tethered agonists, but not the aGPCR GPR110/ADGRF1, M3 muscarinic acetylcholine, or β2 adrenergic GPCRs. DHM inhibited tethered peptide agonist-stimulated and synthetic peptide agonist-stimulated GPR56 but did not inhibit basal activity, demonstrating that it antagonizes the peptide agonist. DHM is a novel aGPCR antagonist and potentially useful chemical probe that may be developed as a future aGPCR therapeutic.

Mitochondrial respiration results in an electrochemical proton gradient, or protonmotive force (pmf), across the mitochondrial inner membrane (IM). The pmf is a form of potential energy consisting of charge (Δψ(m)) and chemical (ΔpH) components, that together drive ATP production. In a process called uncoupling, proton leak into the mitochondrial matrix independent of ATP production dissipates the pmf and energy is lost as heat. Other events can directly dissipate the pmf independent of ATP production as well, such as chemical exposure or mechanisms involving regulated mitochondrial membrane electrolyte transport. Uncoupling has defined roles in metabolic plasticity and can be linked through signal transduction to physiologic events. In the latter case, the pmf impacts mitochondrial reactive oxygen species (ROS) production. Though capable of molecular damage, ROS also have signaling properties that depend on the timing, location, and quantity of their production. In this review we provide a general overview of mitochondrial ROS production, mechanisms of uncoupling, and how these work in tandem to affect physiology and pathologies, including: obesity, cardiovascular disease, and immunity. Overall, we highlight that isolated bioenergetic models—mitochondria and cells—only partially recapitulate the complex link between the pmf and ROS signaling that occurs in vivo.

Exercise is a robust stimulus for mitochondrial adaptations in skeletal muscle which consequently plays a central role in enhancing metabolic health. Despite this, the precise molecular events that underpin these beneficial effects remain elusive. In this review, we discuss molecular signals generated during exercise leading to altered mitochondrial morphology and dynamics. In particular, we focus on the interdependence between reactive oxygen species (ROS) and redox homeostasis, the sensing of cellular bioenergetic status via 5’ adenosine monophosphate (AMP)-activated protein kinase (AMPK), and the regulation of mitochondrial fission and fusion. Precisely how exercise regulates the network of these responses and their effects on mitochondrial dynamics is not fully understood at present. We highlight the limitations that exist with the techniques currently available, and discuss novel molecular tools to potentially advance the fields of redox biology and mitochondrial bioenergetics. Ultimately, a greater understanding of these processes may lead to novel mitochondria-targeted therapeutic strategies to augment or mimic exercise in order to attenuate or reverse pathophysiology.

Mitochondria play critical roles in meeting cellular energy demand, in cell death, and in reactive oxygen species (ROS) and stress signaling. Most Caenorhabditis elegans loss-of-function (lf) mutants in nuclear-encoded components of the respiratory chain are non-viable, emphasizing the importance of respiratory function. Chromophore-Assisted Light Inactivation (CALI) using genetically-encoded photosensitizers provides an opportunity to determine how individual respiratory chain components contribute to physiology following acute lf. As proof-of-concept, we expressed the ‘singlet oxygen generator’ miniSOG as a fusion with the SDHC subunit of respiratory complex II, encoded by mev-1 in C. elegans, using Mos1-mediated Single Copy Insertion. The resulting mev-1::miniSOG transgene complemented mev-1 mutant phenotypes in kn1 missense and tm1081(lf) deletion mutants. Complex II activity was inactivated by blue light in mitochondria from strains expressing active miniSOG fusions, but not those from inactive fusions. Moreover, light-inducible phenotypes in vivo demonstrated that complex II activity is important under conditions of high energy demand, and that specific cell types are uniquely susceptible to loss of complex II. In conclusion, miniSOG-mediated CALI is a novel genetic platform for acute inactivation of respiratory chain components. Spatio-temporally controlled ROS generation will expand our understanding of how the respiratory chain and mitochondrial ROS influence whole organism physiology.

Rationale: Tissue ischemia/reperfusion (IR) injury underlies several leading causes of death such as heart-attack and stroke. The lack of clinical therapies for IR injury may be partly due to the difficulty of adapting IR injury models to high-throughput screening (HTS). Objective: To develop a model of IR injury that is both physiologically relevant and amenable to HTS. Methods and Results: A microplate-based respirometry apparatus was used. Controlling gas flow in the plate head space, coupled with the instrument's mechanical systems, yielded a 24-well model of IR injury in which H9c2 cardiomyocytes were transiently trapped in a small volume, rendering them ischemic. After initial validation with known protective molecules, the model was used to screen a 2000-molecule library, with post-IR cell death as an end point. P o 2 and pH monitoring in each well also afforded metabolic data. Ten protective, detrimental, and inert molecules from the screen were subsequently tested in a Langendorff-perfused heart model of IR injury, revealing strong correlations between the screening end point and both recovery of cardiac function (negative, r 2 =0.66) and infarct size (positive, r 2 =0.62). Relationships between the effects of added molecules on cellular bioenergetics and protection against IR injury were also studied. Conclusions: This novel cell-based assay can predict either protective or detrimental effects on IR injury in the intact heart. Its application may help identify therapeutic or harmful molecules.

Hypoxic preconditioning (HP) is an evolutionarily-conserved mechanism that protects an organism against stress. The mitochondrial ATP-sensitive K(+) channel (mK(ATP)) plays an essential role in the protective signaling, but remains molecularly undefined. Several lines of evidence suggest that mK(ATP) may arise from an inward rectifying K(+) channel (Kir). The genetic model organism Caenorhabditis elegans exhibits HP and displays mK(ATP) activity. Here, we investigate the tissue expression profile of the three C. elegans Kir genes and demonstrate that mutant strains where the irk genes have been deleted either individually or in combination can be protected by HP and exhibit robust mK(ATP) channel activity in purified mitochondria. These data suggest that the mK(ATP) in C. elegans does not arise from a Kir derived channel.

While reactive oxygen species (ROS) are known to have a harmful role within a cell, ROS can also have a protective signaling role. One limitation to differentiating these roles is the inability to spatially and temporally control ROS production. ROS-generating proteins (RGPs) overcome this limitation by using light to control ROS production. RGPs have been used in a variety of applications, from cell ablation to mutagenesis; however, the type of ROS produced is often not well characterized. One RGP, mini “Singlet Oxygen Generator” was reported to generate large quantities of singlet oxygen. However, the contribution of superoxide remains unclear due to indirect measures with confounding interpretations. We measured the light-dependent superoxide production of purified miniSOG using HPLC separation of dihydroethidium oxidation products. We demonstrate that miniSOG generates superoxide in an SOD-dependent and azide/catalase-independent manner. miniSOG’s superoxide production rate was lower than its free chromophore, flavin mononucleotide. miniSOG produced superoxide at a rate of ~4.0 µmol superoxide/min/µmol photosensitizer for an excitation fluence rate of 5.9 mW/mm2 at 470±20 nm, and the rate remained consistent across fluences. This contrasts miniSOG’s singlet oxygen production which has been shown to be fluence-dependent. Understanding the type and amount of biologically available ROS produced by miniSOG can improve the experimental design and interpretation of the results. Our results may aid in the design of new RGPs that can be more selective in their photosensitization mechanism.

Mitochondria use an electrochemical proton gradient to produce ATP, the main cellular energy source. This gradient, called the protonmotive force (PMF), also controls substrate uptake, calcium signaling, and reactive oxygen species (ROS) production and signaling. Despite the central role of the PMF in mitochondrial bioenergetics, current tools to modulate the PMF lack spatial and temporal control in vivo. For example, protonophores are used to irreversibly decrease the PMF, but this pharmacologic approach lacks tissue and mitochondria-selectivity. Importantly, there are no means to increase the PMF. Here, we characterize an optogenetic approach to selectively increase the PMF and affect mitochondrial function in vivo with light. Using a novel mitochondria-targeting strategy we expressed a light-activated proton pump in mitochondria of Caenorhabditis elegans. In response to light, the mitochondria-targeted light-activated proton pump (mtLAPP) increased the PMF in isolated mitochondria and whole animals, observed through tetramethylrhodamine ethyl ester fluorescence. Additionally, optogenetic stimulation of bioenergetics by mtLAPP can overcome toxicity from inhibition of proton-pumping complexes of the mitochondrial electron transport chain. Our results provide a novel method to enhance mitochondrial bioenergetics in whole organisms, and may be used to affect physiologic outputs downstream of the PMF, such as ROS signaling and metabolism.

The tumor microenvironment presents significant metabolic challenges to T cells by depleting oxygen and glucose, as well as limiting the uptake of key nutrients. Therefore, T cells and tumor cells engage in fierce metabolic competition, as the demand for both oxygen and glucose in the niche is extremely high. The transition from a resting naïve T cell into an activated and highly proliferative effector T cell requires substantial metabolic reprogramming from relying primarily on oxidative phosphorylation (OxPhos) to the rapid induction of aerobic glycolysis. However, evidence suggests that tumor infiltrating CD8+ T cells show defects in glycolytic functions. In addition, our data indicates that actively migrating effector CD8+ T cells have greater levels of OxPhos activity than stationary cells, imposing an increasing demand for oxygen during T cell migration to the tumor site. To overcome the glycolytic deficiency of the tumor-targeting T cells and boost anti-tumor effector functions at the tumor microenvironment, we developed a genetically encoded light-activated proton pump (fungal proton pump, “Mac”), namely photoactivatable OxPhos (PA-OxPhos) that is expressed in the inner mitochondrial membrane. During OxPhos, electrons enter the electron transport chain (ETC), causing protons to be pumped across the inner mitochondrial membrane to establish a proton gradient. The gradient is then used to generate ATP through complex V (CxV). Therefore, the outward proton pumping through the inner mitochondrial membrane by light stimulation of PA-OxPhos mimics the ETC function and boosts ATP generation in T cells, even in the presence of low levels of oxygen and substrates, giving T cells a metabolic competitive advantage in the tumor microenvironment. PA-OxPhos is tagged with GFP and is expressed in the mitochondria of transfected 293T cells and in activated mouse CD8+ T cells. When cells were treated with Rotenone (an inhibitor of complex I of the ETC), ATP production was decreased after 90 minutes. Importantly, light stimulation of 293T cells expressing PA-OxPhos successfully increased ATP production even in the presence of Rotenone. Our data suggests that PA-OxPhos can remotely provide a competitive metabolic advantage and boost T cell functions in the tumor microenvironment. The utilization of an alternative mechanism for ATP production in T cells could potentially dissipate the failures of T-cell-based cancer immunotherapies in destroying malignant cells of solid tumors. Citation Format: Andrea Amitrano, Brandon Walling, Kyun Do Kim, Brandon Berry, Adam Trewin, Andrew Wojtovich, Minsoo Kim. Optogenetic regulation of T cell metabolism in the tumor microenvironment [abstract]. In: Proceedings of the AACR Special Conference on Tumor Immunology and Immunotherapy; 2017 Oct 1-4; Boston, MA. Philadelphia (PA): AACR; Cancer Immunol Res 2018;6(9 Suppl):Abstract nr A73.

Most cells have the inherent capacity to shift their reliance on glycolysis relative to oxidative metabolism, and studies in model systems have shown that targeting such shifts may be useful in treating or preventing a variety of diseases ranging from cancer to ischemic injury. However, we currently have a limited number of mechanistically distinct classes of drugs that alter the relative activities of these two pathways. We screen for such compounds by scoring the ability of >3,500 small molecules to selectively impair growth and viability of human fibroblasts in media containing either galactose or glucose as the sole sugar source. We identify several clinically used drugs never linked to energy metabolism, including the antiemetic meclizine, which attenuates mitochondrial respiration through a mechanism distinct from that of canonical inhibitors. We further show that meclizine pretreatment confers cardioprotection and neuroprotection against ischemia-reperfusion injury in murine models. Nutrient-sensitized screening may provide a useful framework for understanding gene function and drug action within the context of energy metabolism.

Mitochondrial reactive oxygen species (ROS) generation and the attendant mitochondrial dysfunction are implicated in a range of disease states. The objective of the present studies was to test the hypothesis that the mitochondrial beta-oxidation pathway could be exploited to deliver and biotransform the prodrugs omega-(phenoxy)alkanoic acids, 3-(phenoxy)acrylic acids, and omega-(1-methyl-1H-imidazol-2-ylthio)alkanoic acids to the corresponding phenolic antioxidants or methimazole. 3- and 5-(Phenoxy)alkanoic acids and methyl-substituted analogs were biotransformed to phenols; rates of biotransformation decreased markedly with methyl-group substitution on the phenoxy moiety. 2,6-Dimethylphenol formation from the analogs 3-([2,6-dimethylphenoxy]methylthio)propanoic acid and 3-(2,6-dimethylphenoxy)acrylic acid was greater than that observed with omega-(2,6-dimethylphenoxy)alkanoic acids. 3- and 5-(1-Methyl-1H-imidazol-2-ylthio)alkanoic acids were rapidly biotransformed to the antioxidant methimazole and conferred significant cytoprotection against hypoxia-reoxygenation injury in isolated cardiomyocytes. Both 3-(2,6-dimethylphenoxy)propanoic acid and 3-(2,6-dimethylphenoxy)acrylic acid also afforded cytoprotection against hypoxia-reoxygenation injury in isolated cardiomyocytes. These results demonstrate that mitochondrial beta-oxidation is a potentially useful delivery system for targeting antioxidants to mitochondria.

The cardioprotective effects of ischemic preconditioning (IPC) can be mimicked or blocked by pharmacologic agents, which modulate the mitochondrial ATP-sensitive potassium (mK(ATP)) channel, thereby implicating this channel in the mechanism of IPC. Cardioprotection can also be achieved via inhibition of mitochondrial respiratory complex II, and significant pharmacologic overlap exists between complex II inhibitors and mK(ATP) channel agonists. However, the relationship between complex II and the mK(ATP) channel remains unclear. Atpenin A5 (AA5) is a potent and specific complex II inhibitor, and herein we report that AA5 (1 nM) also activates the mK(ATP) channel and protects against simulated ischemia-reperfusion (IR) injury in isolated cardiomyocytes. Similar to known mK(ATP) agonists, AA5-mediated protection was sensitive to the mK(ATP) antagonists 5-hydroxydecanoate (5HD) and glyburide. Notably, the optimal mK(ATP) opening and protective concentration of AA5 had no effect on complex II enzymatic activity, suggesting an interaction of AA5 with complex II, but not inhibition of the complex per se, is necessary for protection. A cardioprotective effect of AA5 was also observed in isolated perfused hearts, wherein AA5 increased post-IR contractile function and decreased infarct size, in a 5HD-sensitive manner. In conclusion, the specific complex II inhibitor AA5 is the most potent mK(ATP) activator discovered to date, and provides a novel method of activating mK(ATP) channels and protecting the heart from IR injury.

Ischemic preconditioning (IPC) is an evolutionarily conserved endogenous mechanism whereby short periods of non-lethal exposure to hypoxia alleviate damage caused by subsequent ischemia reperfusion (IR). Pharmacologic targeting has suggested that the mitochondrial ATP-sensitive potassium channel (mK(ATP)) is central to IPC signaling, despite its lack of molecular identity. Here, we report that isolated Caenorhabditis elegans mitochondria have a K(ATP) channel with the same physiologic and pharmacologic characteristics as the vertebrate channel. Since C. elegans also exhibit IPC, our observations provide a framework to study the role of mK(ATP) in IR injury in a genetic model organism.

BackgroundAnesthetic preconditioning (APC) is a clinically important phenomenon in which volatile anesthetics (VAs) protect tissues such as heart against ischemic injury. The mechanism of APC is thought to involve K+ channels encoded by the Slo gene family, and the authors showed previously that slo-2 is required for APC in Caenorhabditis elegans. Thus, the authors hypothesized that a slo-2 ortholog may mediate APC-induced cardioprotection in mammals.MethodsA perfused heart model of ischemia–reperfusion injury, a fluorescent assay for K+ flux, and mice lacking Slo2.1 (Slick), Slo2.2 (Slack), or both (double knockouts, Slo2.x dKO) were used to test whether these channels are required for APC-induced cardioprotection and for cardiomyocyte or mitochondrial K+ transport.ResultsIn wild-type (WT) hearts, APC improved post-ischemia–reperfusion functional recovery (APC = 39.5 ± 3.7% of preischemic rate × pressure product vs. 20.3 ± 2.3% in controls, means ± SEM, P = 0.00051, unpaired two-tailed t test, n = 8) and lowered infarct size (APC = 29.0 ± 4.8% of LV area vs. 51.4 ± 4.5% in controls, P = 0.0043, n = 8). Protection by APC was absent in hearts from Slo2.1−/− mice (% recovery APC = 14.6 ± 2.6% vs. 16.5 ± 2.1% in controls, P = 0.569, n = 8 to 9, infarct APC = 52.2 ± 5.4% vs. 53.5 ± 4.7% in controls, P = 0.865, n = 8 to 9). APC protection was also absent in Slo2.x dKO hearts (% recovery APC = 11.0 ± 1.7% vs. 11.9 ± 2.2% in controls, P = 0.725, n = 8, infarct APC = 51.6 ± 4.4% vs. 50.5 ± 3.9% in controls, P = 0.855, n = 8). Meanwhile, Slo2.2−/− hearts responded similar to WT (% recovery APC = 41.9 ± 4.0% vs. 18.0 ± 2.5% in controls, P = 0.00016, n = 8, infarct APC = 25.2 ± 1.3% vs. 50.8 ± 3.3% in controls, P < 0.000005, n = 8). Furthermore, VA-stimulated K+ transport seen in cardiomyocytes or mitochondria from WT or Slo2.2−/− mice was absent in Slo2.1−/− or Slo2.x dKO.ConclusionSlick (Slo2.1) is required for both VA-stimulated K+ flux and for the APC-induced cardioprotection.

A major challenge in drug discovery is to distinguish the molecular targets of a bioactive compound from the hundreds to thousands of additional gene products that respond indirectly to changes in the activity of the targets1,2,3,4,5,6,7,8. Here, we present an integrated computational-experimental approach for computing the likelihood that gene products and associated pathways are targets of a compound. This is achieved by filtering the mRNA expression profile of compound-exposed cells using a reverse-engineered model of the cell's gene regulatory network. We apply the method to a set of 515 whole-genome yeast expression profiles resulting from a variety of treatments (compounds, knockouts and induced expression), and correctly enrich for the known targets and associated pathways in the majority of compounds examined. We demonstrate our approach with PTSB, a growth inhibitory compound with a previously unknown mode of action, by predicting and validating thioredoxin and thioredoxin reductase as its target.

Mitochondrial reactive oxygen species (mtROS) are associated with disease but also required for redox signaling and maintaining cellular homoeostasis. Whether mtROS have beneficial or deleterious effects may depend on the subcellular microdomain. However, to date, the tools available to investigate ROS-specific effects in vivo lack the precise spatial and temporal control. To address this, we expressed the optogenetic tool supernova, a genetically encoded light-activated ROS generating protein, in C. elegans using CRISPR/Cas9. Supernova was fused to mitochondrial complex-II subunits such that it produced ROS in the matrix (SDHB-1::SN) or the intermembrane space (SDHC-1::SN). Localization was confirmed via confocal microscopy and proteinase-K protection assays. To assess redox signaling responses to ROS in these microdomains, we used a GFP transcriptional reporter strain for SKN-1/Nrf-2. Worms were pre-treated with light (5 hrs, 540-590 nm, 2 mW/mm2), then 16 hrs later GFP expression was assessed. GFP increased in SDHC-1::SN but not in SDHB-1::SN worms (p In conclusion, localized matrix ROS generation may lead to oxidative damage, whereas intermembrane space ROS generation induces redox signaling. This suggests that the physiologic output of an ROS microdomain depends on membrane barriers and the compartmentalization of redox sensitive targets.

Introduction: Volatile anesthetic preconditioning (APC) protects the heart from ischemia-reperfusion (IR) injury. APC elicits evolutionarily-conserved protective signaling pathways that converge at the mitochondrial level, where the Slo family of K + channels is thought to mediate protection through an unknown mechanism. Recent work in C. elegans has focused attention on the Slo2 gene product as a transducer of APC effects on hypoxic survival. In mammals, Slo2 has diverged into two paralogs, Slo 2.1 (KCNT2; Slick) and Slo2.2 (KCNT1; Slack). These genes code for Na + -activated K + channels and are highly expressed in brain, but their function in cardiomyocytes and/or mitochondria is unknown. Methods: The contribution of Slo channels to cardiac physiology was characterized using knockout mice, including Slo1 and two novel Slo2.x alleles. APC was assessed through ex-vivo cardiac IR injury. Isolated mitochondrial K + channel activity was assessed using a Tl + flux assay, and by patch-clamp of cardiac mitoplasts. Electron microscopy was used to assess mitochondrial morphology in primary cardiomyocytes and Seahorse extracellular flux analysis used to assess bioenergetics. Results: The Slo2.x ( double KO ) and the Slo2.1 single KO mice could not be protected from cardiac IR injury by APC. Physiologic approaches demonstrated that Slo2.1 is present in mitochondria and Slo2.1 -dependent mitochondrial K + transport can be triggered directly by volatile anesthetics. Cardiomyocytes from Slo2.x dKO mice exhibited profound metabolic remodeling and electron microscopy revealed that Slo2.1 knockouts had enlarged circular mitochondria. In contrast, Slo1 KO mice responded normally to APC and exhibited wild type mitochondrial physiology. Conclusion: Slo2.1 activation protects against cardiac IR, and is required for APC. Slo2.1 also contributes to mitochondrial metabolic homeostasis. As a molecular target for APC, identification of Slo2.1 may facilitate development of targeted therapeutic molecules that can protect the heart and minimize the side effects of volatile anesthetics.

ATP-regulated potassium (KATP) channel complexes of inward rectifier potassium channel (Kir) 6.2 and sulfonylurea receptor (SUR) 1 critically regulate pancreatic islet β-cell membrane potential, calcium influx, and insulin secretion, and consequently, represent important drug targets for metabolic disorders of glucose homeostasis. The KATP channel opener diazoxide is used clinically to treat intractable hypoglycemia caused by excessive insulin secretion, but its use is limited by off-target effects due to lack of potency and selectivity. Some progress has been made in developing improved Kir6.2/SUR1 agonists from existing chemical scaffolds and compound screening, but there are surprisingly few distinct chemotypes that are specific for SUR1-containing KATP channels. Here we report the serendipitous discovery in a high-throughput screen of a novel activator of Kir6.2/SUR1: VU0071063 [7-(4-(tert-butyl)benzyl)-1,3-dimethyl-1H-purine-2,6(3H,7H)-dione]. The xanthine derivative rapidly and dose-dependently activates Kir6.2/SUR1 with a half-effective concentration (EC50) of approximately 7 μM, is more efficacious than diazoxide at low micromolar concentrations, directly activates the channel in excised membrane patches, and is selective for SUR1- over SUR2A-containing Kir6.1 or Kir6.2 channels, as well as Kir2.1, Kir2.2, Kir2.3, Kir3.1/3.2, and voltage-gated potassium channel 2.1. Finally, we show that VU0071063 activates native Kir6.2/SUR1 channels, thereby inhibiting glucose-stimulated calcium entry in isolated mouse pancreatic β cells. VU0071063 represents a novel tool/compound for investigating β-cell physiology, KATP channel gating, and a new chemical scaffold for developing improved activators with medicinal chemistry.