Using light to drive energy transduction in metazoan aging.

Mitochondrial dysfunction is a central hallmark of aging and energy transduction is a promising target for longevity interventions. New research suggests that interventions in how energy is transduced could benefit healthy longevity. Here, we propose using light as an alternative energy source to fuel mitochondria and increase metazoan lifespan.

Optogenetic control of mitochondrial protonmotive force to impact cellular stress resistance.

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.

Exploratory Analysis of Associations Between Whole Blood Mitochondrial Gene Expression and Cancer-Related Fatigue Among Breast Cancer Survivors.

BACKGROUND: Cancer-related fatigue is a prevalent, debilitating, and persistent condition. Mitochondrial dysfunction is a putative contributor to cancer-related fatigue, but relationships between mitochondrial function and cancer-related fatigue are not well understood. OBJECTIVES: We investigated the relationships between mitochondrial DNA (mtDNA) gene expression and cancer-related fatigue, as well as the effects of fish and soybean oil supplementation on these relationships. METHODS: A secondary analysis was performed on data from a randomized controlled trial of breast cancer survivors 4-36 months posttreatment with moderate-severe cancer-related fatigue. Participants were randomized to take 6 g fish oil, 6 g soybean oil, or 3 g each daily for 6 weeks. At pre- and postintervention, participants completed the Functional Assessment of Chronic Illness Therapy-Fatigue questionnaire and provided whole blood for assessment of mtDNA gene expression. The expression of 12 protein-encoding genes was reduced to a single dimension using principal component analysis for use in regression analysis. Relationships between mtDNA expression and cancer-related fatigue were assessed using linear regression. RESULTS: Among 68 participants, cancer-related fatigue improved and expression of all mtDNA genes decreased over 6 weeks with no effect of treatment group on either outcome. Participants with lower baseline mtDNA gene expression had greater improvements in cancer-related fatigue. No significant associations were observed between mtDNA gene expression and cancer-related fatigue at baseline or changes in mtDNA gene expression and changes in cancer-related fatigue. DISCUSSION: Data from this exploratory study add to the growing literature that mitochondrial dysfunction may contribute to the etiology and pathophysiology of cancer-related fatigue.

Neuronal AMPK coordinates mitochondrial energy sensing and hypoxia resistance in C. elegans.

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.

Dihydromunduletone Is a Small-Molecule Selective Adhesion G Protein-Coupled Receptor Antagonist.

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 ß2 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.

Cardiac Slo2.1 Is Required for Volatile Anesthetic Stimulation of K+ Transport and Anesthetic Preconditioning.

BACKGROUND: Anesthetic 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. METHODS: A 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. RESULTS: In 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. CONCLUSION: Slick (Slo2.1) is required for both VA-stimulated K flux and for the APC-induced cardioprotection.

Direct activation of ß-cell KATP channels with a novel xanthine derivative.

ATP-regulated potassium (KATP) channel complexes of inward rectifier potassium channel (Kir) 6.2 and sulfonylurea receptor (SUR) 1 critically regulate pancreatic islet ß-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 ß cells. VU0071063 represents a novel tool/compound for investigating ß-cell physiology, KATP channel gating, and a new chemical scaffold for developing improved activators with medicinal chemistry.

Physiological consequences of complex II inhibition for aging, disease, and the mKATP channel.

In recent years, it has become apparent that there exist several roles for respiratory complex II beyond metabolism. These include: (i) succinate signaling, (ii) reactive oxygen species (ROS) generation, (iii) ischemic preconditioning, (iv) various disease states and aging, and (v) a role in the function of the mitochondrial ATP-sensitive K(+) (mKATP) channel. This review will address the involvement of complex II in each of these areas, with a focus on how complex II regulates or may be involved in the assembly of the mKATP. This article is part of a Special Issue entitled: Respiratory complex II: Role in cellular physiology and disease.

A cell-based phenotypic assay to identify cardioprotective agents.

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. Po2 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, r2=0.66) and infarct size (positive, r2=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.

DMT1 knockout abolishes ferroptosis induced mitochondrial dysfunction in C. elegans amyloid ß proteotoxicity.

Iron is critical for neuronal activity and metabolism, and iron dysregulation alters these functions in age-related neurodegenerative disorders, such as Alzheimer's disease (AD). AD is a chronic neurodegenerative disease characterized by progressive neuronal dysfunction, memory loss and decreased cognitive function. AD patients exhibit elevated iron levels in the brain compared to age-matched non-AD individuals. However, the degree to which iron overload contributes to AD pathogenesis is unclear. Here, we evaluated the involvement of ferroptosis, an iron-dependent cell death process, in mediating AD-like pathologies in C. elegans. Results showed that iron accumulation occurred prior to the loss of neuronal function as worms age. In addition, energetic imbalance was an early event in iron-induced loss of neuronal function. Furthermore, the loss of neuronal function was, in part, due to increased mitochondrial reactive oxygen species mediated oxidative damage, ultimately resulting in ferroptotic cell death. The mitochondrial redox environment and ferroptosis were modulated by pharmacologic processes that exacerbate or abolish iron accumulation both in wild-type worms and worms with increased levels of neuronal amyloid beta (Aß). However, neuronal Aß worms were more sensitive to ferroptosis-mediated neuronal loss, and this increased toxicity was ameliorated by limiting the uptake of ferrous iron through knockout of divalent metal transporter 1 (DMT1). In addition, DMT1 knockout completely suppressed phenotypic measures of Aß toxicity with age. Overall, our findings suggest that iron-induced ferroptosis alters the mitochondrial redox environment to drive oxidative damage when neuronal Aß is overexpressed. DMT1 knockout abolishes neuronal Aß-associated pathologies by reducing neuronal iron uptake.