Mitochondrial respiratory-chain adaptations in macrophages contribute to antibacterial host defense.

Macrophages tightly scale their core metabolism after being activated, but the precise regulation of the mitochondrial electron-transport chain (ETC) and its functional implications are currently unknown. Here we found that recognition of live bacteria by macrophages transiently decreased assembly of the ETC complex I (CI) and CI-containing super-complexes and switched the relative contributions of CI and CII to mitochondrial respiration. This was mediated by phagosomal NADPH oxidase and the reactive oxygen species (ROS)-dependent tyrosine kinase Fgr. It required Toll-like receptor signaling and the NLRP3 inflammasome, which were both connected to bacterial viability-specific immune responses. Inhibition of CII during infection with Escherichia coli normalized serum concentrations of interleukin 1ß (IL-1ß) and IL-10 to those in mice treated with dead bacteria and impaired control of bacteria. We have thus identified ETC adaptations as an early immunological-metabolic checkpoint that adjusts innate immune responses to bacterial infection.

Inhibition of ATP synthase reverse activity restores energy homeostasis in mitochondrial pathologies.

The maintenance of cellular function relies on the close regulation of adenosine triphosphate (ATP) synthesis and hydrolysis. ATP hydrolysis by mitochondrial ATP Synthase (CV) is induced by loss of proton motive force and inhibited by the mitochondrial protein ATPase inhibitor (ATPIF1). The extent of CV hydrolytic activity and its impact on cellular energetics remains unknown due to the lack of selective hydrolysis inhibitors of CV. We find that CV hydrolytic activity takes place in coupled intact mitochondria and is increased by respiratory chain defects. We identified (+)-Epicatechin as a selective inhibitor of ATP hydrolysis that binds CV while preventing the binding of ATPIF1. In cells with Complex-III deficiency, we show that inhibition of CV hydrolytic activity by (+)-Epichatechin is sufficient to restore ATP content without restoring respiratory function. Inhibition of CV-ATP hydrolysis in a mouse model of Duchenne Muscular Dystrophy is sufficient to improve muscle force without any increase in mitochondrial content. We conclude that the impact of compromised mitochondrial respiration can be lessened using hydrolysis-selective inhibitors of CV.

Na+ controls hypoxic signalling by the mitochondrial respiratory chain.

All metazoans depend on the consumption of O2 by the mitochondrial oxidative phosphorylation system (OXPHOS) to produce energy. In addition, the OXPHOS uses O2 to produce reactive oxygen species that can drive cell adaptations1-4, a phenomenon that occurs in hypoxia4-8 and whose precise mechanism remains unknown. Ca2+ is the best known ion that acts as a second messenger9, yet the role ascribed to Na+ is to serve as a mere mediator of membrane potential10. Here we show that Na+ acts as a second messenger that regulates OXPHOS function and the production of reactive oxygen species by modulating the fluidity of the inner mitochondrial membrane. A conformational shift in mitochondrial complex I during acute hypoxia11 drives acidification of the matrix and the release of free Ca2+ from calcium phosphate (CaP) precipitates. The concomitant activation of the mitochondrial Na+/Ca2+ exchanger promotes the import of Na+ into the matrix. Na+ interacts with phospholipids, reducing inner mitochondrial membrane fluidity and the mobility of free ubiquinone between complex II and complex III, but not inside supercomplexes. As a consequence, superoxide is produced at complex III. The inhibition of Na+ import through the Na+/Ca2+ exchanger is sufficient to block this pathway, preventing adaptation to hypoxia. These results reveal that Na+ controls OXPHOS function and redox signalling through an unexpected interaction with phospholipids, with profound consequences for cellular metabolism.

A novel approach to measure mitochondrial respiration in frozen biological samples.

Respirometry is the gold standard measurement of mitochondrial oxidative function, as it reflects the activity of the electron transport chain complexes working together. However, the requirement for freshly isolated mitochondria hinders the feasibility of respirometry in multi-site clinical studies and retrospective studies. Here, we describe a novel respirometry approach suited for frozen samples by restoring electron transfer components lost during freeze/thaw and correcting for variable permeabilization of mitochondrial membranes. This approach preserves 90-95% of the maximal respiratory capacity in frozen samples and can be applied to isolated mitochondria, permeabilized cells, and tissue homogenates with high sensitivity. We find that primary changes in mitochondrial function, detected in fresh tissue, are preserved in frozen samples years after collection. This approach will enable analysis of the integrated function of mitochondrial Complexes I to IV in one measurement, collected at remote sites or retrospectively in samples residing in tissue biobanks.

p38γ and p38δ regulate postnatal cardiac metabolism through glycogen synthase 1.

During the first weeks of postnatal heart development, cardiomyocytes undergo a major adaptive metabolic shift from glycolytic energy production to fatty acid oxidation. This metabolic change is contemporaneous to the up-regulation and activation of the p38γ and p38δ stress-activated protein kinases in the heart. We demonstrate that p38γ/δ contribute to the early postnatal cardiac metabolic switch through inhibitory phosphorylation of glycogen synthase 1 (GYS1) and glycogen metabolism inactivation. Premature induction of p38γ/δ activation in cardiomyocytes of newborn mice results in an early GYS1 phosphorylation and inhibition of cardiac glycogen production, triggering an early metabolic shift that induces a deficit in cardiomyocyte fuel supply, leading to whole-body metabolic deregulation and maladaptive cardiac pathogenesis. Notably, the adverse effects of forced premature cardiac p38γ/δ activation in neonate mice are prevented by maternal diet supplementation of fatty acids during pregnancy and lactation. These results suggest that diet interventions have a potential for treating human cardiac genetic diseases that affect heart metabolism.

Heteroplasmy of Wild-Type Mitochondrial DNA Variants in Mice Causes Metabolic Heart Disease With Pulmonary Hypertension and Frailty.

BACKGROUND: In most eukaryotic cells, the mitochondrial DNA (mtDNA) is transmitted uniparentally and present in multiple copies derived from the clonal expansion of maternally inherited mtDNA. All copies are therefore near-identical, or homoplasmic. The presence of >1 mtDNA variant in the same cytoplasm can arise naturally or result from new medical technologies aimed at preventing mitochondrial genetic diseases and improving fertility. The latter is called divergent nonpathologic mtDNA heteroplasmy (DNPH). We hypothesized that DNPH is maladaptive and usually prevented by the cell. METHODS: We engineered and characterized DNPH mice throughout their lifespan using transcriptomic, metabolomic, biochemical, physiologic, and phenotyping techniques. We focused on in vivo imaging techniques for noninvasive assessment of cardiac and pulmonary energy metabolism. RESULTS: We show that DNPH impairs mitochondrial function, with profound consequences in critical tissues that cannot resolve heteroplasmy, particularly cardiac and skeletal muscle. Progressive metabolic stress in these tissues leads to severe pathology in adulthood, including pulmonary hypertension and heart failure, skeletal muscle wasting, frailty, and premature death. Symptom severity is strongly modulated by the nuclear context. CONCLUSIONS: Medical interventions that may generate DNPH should address potential incompatibilities between donor and recipient mtDNA.

The Potential Role of Epigallocatechin-3-Gallate (EGCG) in Breast Cancer Treatment.

Breast cancer is one of the most diagnosed cancers worldwide, with an incidence of 47.8%. Its treatment includes surgery, radiotherapy, chemotherapy, and antibodies giving a mortality of 13.6%. Breast tumor development is driven by a variety of signaling pathways with high heterogeneity of surface receptors, which makes treatment difficult. Epigallocatechin-3-gallate (EGCG) is a natural polyphenol isolated as the main component in green tea; it has shown multiple beneficial effects in breast cancer, controlling proliferation, invasion, apoptosis, inflammation, and demethylation of DNA. These properties were proved in vitro and in vivo together with synergistic effects in combination with traditional chemotherapy, increasing the effectiveness of the treatment. This review focuses on the effects of EGCG on the functional capabilities acquired by breast tumor cells during its multistep development, the molecular and signal pathways involved, the synergistic effects in combination with current drugs, and how nanomaterials can improve its bioavailability on breast cancer treatment.

Natural Compounds Purified from the Leaves of Aristotelia chilensis: Makomakinol, a New Alkaloid and the Effect of Aristoteline and Hobartine on NaV Channels.

Aristotelia chilensis or "maqui" is a tree native to Chile used in the folk medicine of the Mapuche people as an anti-inflammatory agent for the treatment of digestive ailments, fever, and skin lesions. Maqui fruits are black berries which are considered a "superfruit" with notable potential health benefits, promoted to be an antioxidant, cardioprotective, and anti-inflammatory. Maqui leaves contain non-iridoid monoterpene indole alkaloids which have previously been shown to act on nicotinic acetylcholine receptors, potassium channels, and calcium channels. Here, we isolated a new alkaloid from maqui leaves, now called makomakinol, together with the known alkaloids aristoteline, hobartine, and 3-formylindole. Moreover, the polyphenols quercetine, ethyl caffeate, and the terpenes, dihydro-ß-ionone and terpin hydrate, were also obtained. In light of the reported analgesic and anti-nociceptive properties of A. chilensis, in particular a crude mixture of alkaloids containing aristoteline and hobartinol (PMID 21585384), we therefore evaluated the activity of aristoteline and hobartine on NaV1.8, a key NaV isoform involved in nociception, using automated whole-cell patch-clamp electrophysiology. Aristoteline and hobartine both inhibited Nav1.8 with an IC50 of 68 ± 3 µM and 54 ± 1 µM, respectively. Hobartine caused a hyperpolarizing shift of the voltage-dependence of the activation, whereas aristoteline did not change the voltage-dependence of the activation or inactivation. The inhibitory activity of these alkaloids on NaV channels may contribute to the reported analgesic properties of Aristotelia chilensis used by the Mapuche people.

Ginkgolides and Huperzine A for complementary treatment of Alzheimer's disease.

Alzheimer's disease (AD) is a neurodegenerative disorder characterized by gradual deterioration of cognitive function, memory, and inability to perform daily, social, or occupational activities. Its etiology is associated with the accumulation of ß-amyloid peptides, phosphorylated tau protein, and neuroinflammatory and oxidative processes in the brain. Currently, there is no successful pharmacological treatment for AD. The few approved drugs are mainly aimed at treating the symptoms; however, due to the increasing discovery of etiopathological factors, there are great efforts to find new multifunctional molecules to slow down the course of this neurodegenerative disease. The commercial Ginkgo biloba formulation EGb 761® and Huperzine A, an alkaloid present in the plant Huperzia serrata, have shown in clinical trials to possess cholinergic and neuroprotective activities, including improvement in cognition, activities of daily living, and neuropsychiatric symptoms in AD patients. The purpose of this review is to expose the positive results of intervention with EGb 761® and Huperzine in patients with mild to moderate AD in the last 10 years, highlighting the pharmacological functions that justify their use in AD therapy.

ATP-consuming futile cycles as energy dissipating mechanisms to counteract obesity.

Obesity results from an imbalance in energy homeostasis, whereby excessive energy intake exceeds caloric expenditure. Energy can be dissipated out of an organism by producing heat (thermogenesis), explaining the long-standing interest in exploiting thermogenic processes to counteract obesity. Mitochondrial uncoupling is a process that expends energy by oxidizing nutrients to produce heat, instead of ATP synthesis. Energy can also be dissipated through mechanisms that do not involve mitochondrial uncoupling. Such mechanisms include futile cycles described as metabolic reactions that consume ATP to produce a product from a substrate but then converting the product back into the original substrate, releasing the energy as heat. Energy dissipation driven by cellular ATP demand can be regulated by adjusting the speed and number of futile cycles. Energy consuming futile cycles that are reviewed here are lipolysis/fatty acid re-esterification cycle, creatine/phosphocreatine cycle, and the SERCA-mediated calcium import and export cycle. Their reliance on ATP emphasizes that mitochondrial oxidative function coupled to ATP synthesis, and not just uncoupling, can play a role in thermogenic energy dissipation. Here, we review ATP consuming futile cycles, the evidence for their function in humans, and their potential employment as a strategy to dissipate energy and counteract obesity.

Blocking mitochondrial pyruvate import in brown adipocytes induces energy wasting via lipid cycling.

Combined fatty acid esterification and lipolysis, termed lipid cycling, is an ATP-consuming process that contributes to energy expenditure. Therefore, interventions that stimulate energy expenditure through lipid cycling are of great interest. Here we find that pharmacological and genetic inhibition of the mitochondrial pyruvate carrier (MPC) in brown adipocytes activates lipid cycling and energy expenditure, even in the absence of adrenergic stimulation. We show that the resulting increase in ATP demand elevates mitochondrial respiration coupled to ATP synthesis and fueled by lipid oxidation. We identify that glutamine consumption and the Malate-Aspartate Shuttle are required for the increase in Energy Expenditure induced by MPC inhibition in Brown Adipocytes (MAShEEBA). We thus demonstrate that energy expenditure through enhanced lipid cycling can be activated in brown adipocytes by decreasing mitochondrial pyruvate availability. We present a new mechanism to increase energy expenditure and fat oxidation in brown adipocytes, which does not require adrenergic stimulation of mitochondrial uncoupling.

Mitochondrial and nuclear DNA matching shapes metabolism and healthy ageing.

Human mitochondrial DNA (mtDNA) shows extensive within population sequence variability. Many studies suggest that mtDNA variants may be associated with ageing or diseases, although mechanistic evidence at the molecular level is lacking. Mitochondrial replacement has the potential to prevent transmission of disease-causing oocyte mtDNA. However, extension of this technology requires a comprehensive understanding of the physiological relevance of mtDNA sequence variability and its match with the nuclear-encoded mitochondrial genes. Studies in conplastic animals allow comparison of individuals with the same nuclear genome but different mtDNA variants, and have provided both supporting and refuting evidence that mtDNA variation influences organismal physiology. However, most of these studies did not confirm the conplastic status, focused on younger animals, and did not investigate the full range of physiological and phenotypic variability likely to be influenced by mitochondria. Here we systematically characterized conplastic mice throughout their lifespan using transcriptomic, proteomic,metabolomic, biochemical, physiological and phenotyping studies. We show that mtDNA haplotype profoundly influences mitochondrial proteostasis and reactive oxygen species generation,insulin signalling, obesity, and ageing parameters including telomere shortening and mitochondrial dysfunction, resulting in profound differences in health longevity between conplastic strains.

Increased localization of APP-C99 in mitochondria-associated ER membranes causes mitochondrial dysfunction in Alzheimer disease.

In the amyloidogenic pathway associated with Alzheimer disease (AD), the amyloid precursor protein (APP) is cleaved by ß-secretase to generate a 99-aa C-terminal fragment (C99) that is then cleaved by γ-secretase to generate the ß-amyloid (Aß) found in senile plaques. In previous reports, we and others have shown that γ-secretase activity is enriched in mitochondria-associated endoplasmic reticulum (ER) membranes (MAM) and that ER-mitochondrial connectivity and MAM function are upregulated in AD We now show that C99, in addition to its localization in endosomes, can also be found in MAM, where it is normally processed rapidly by γ-secretase. In cell models of AD, however, the concentration of unprocessed C99 increases in MAM regions, resulting in elevated sphingolipid turnover and an altered lipid composition of both MAM and mitochondrial membranes. In turn, this change in mitochondrial membrane composition interferes with the proper assembly and activity of mitochondrial respiratory supercomplexes, thereby likely contributing to the bioenergetic defects characteristic of AD.

p38α blocks brown adipose tissue thermogenesis through p38δ inhibition.

Adipose tissue has emerged as an important regulator of whole-body metabolism, and its capacity to dissipate energy in the form of heat has acquired a special relevance in recent years as potential treatment for obesity. In this context, the p38MAPK pathway has arisen as a key player in the thermogenic program because it is required for the activation of brown adipose tissue (BAT) thermogenesis and participates also in the transformation of white adipose tissue (WAT) into BAT-like depot called beige/brite tissue. Here, using mice that are deficient in p38α specifically in adipose tissue (p38αFab-KO), we unexpectedly found that lack of p38α protected against high-fat diet (HFD)-induced obesity. We also showed that p38αFab-KO mice presented higher energy expenditure due to increased BAT thermogenesis. Mechanistically, we found that lack of p38α resulted in the activation of the related protein kinase family member p38δ. Our results showed that p38δ is activated in BAT by cold exposure, and lack of this kinase specifically in adipose tissue (p38δ Fab-KO) resulted in overweight together with reduced energy expenditure and lower body and skin surface temperature in the BAT region. These observations indicate that p38α probably blocks BAT thermogenesis through p38δ inhibition. Consistent with the results obtained in animals, p38α was reduced in visceral and subcutaneous adipose tissue of subjects with obesity and was inversely correlated with body mass index (BMI). Altogether, we have elucidated a mechanism implicated in physiological BAT activation that has potential clinical implications for the treatment of obesity and related diseases such as diabetes.

Halotolerance mechanisms of the methanotroph Methylomicrobium alcaliphilum.

Methylomicrobium alcaliphilum is an alkaliphilic and halotolerant methanotroph. The physiological responses of M. alcaliphilum to high NaCl concentrations, were studied using RNA sequencing and metabolic modeling. This study revealed that M. alcaliphilum possesses an unusual respiratory chain, in which complex I is replaced by a Na+ extruding NQR complex (highly upregulated under high salinity conditions) and a Na+ driven adenosine triphosphate (ATP) synthase coexists with a conventional H+ driven ATP synthase. A thermodynamic and metabolic model showing the interplay between these different components is presented. Ectoine is the main osmoprotector used by the cells. Ectoine synthesis is activated by the transcription of an ect operon that contains five genes, including the ectoine hydroxylase coding ectD gene. Enzymatic tests revealed that the product of ectD does not have catalytic activity. A new Genome Scale Metabolic Model for M. alcaliphilum revealed a higher flux in the oxidative branch of the pentose phosphate pathway leading to NADPH production and contributing to resistance to oxidative stress.

Activation energies for two steps in the S2→ S3 transition of photosynthetic water oxidation from time-resolved single-frequency infrared spectroscopy.

The mechanism of water oxidation by the Photosystem II (PSII) protein-cofactor complex is of high interest, but specifically, the crucial coupling of protonation dynamics to electron transfer (ET) and dioxygen chemistry remains insufficiently understood. We drove spinach-PSII membranes by nanosecond-laser flashes synchronously through the water-oxidation cycle and traced the PSII processes by time-resolved single-frequency infrared (IR) spectroscopy in the spectral range of symmetric carboxylate vibrations of protein side chains. After the collection of IR-transients from 100 ns to 1 s, we analyzed the proton-removal step in the S2 ⇒ S3 transition, which precedes the ET that oxidizes the Mn4CaOx-cluster. Around 1400 cm-1, pronounced changes in the IR-transients reflect this pre-ET process (∼40 µs at 20 °C) and the ET step (∼300 µs at 20 °C). For transients collected at various temperatures, unconstrained multi-exponential simulations did not provide a coherent set of time constants, but constraining the ET time constants to previously determined values solved the parameter correlation problem and resulted in an exceptionally high activation energy of 540 ± 30 meV for the pre-ET step. We assign the pre-ET step to deprotonation of a group that is re-protonated by accepting a proton from the substrate-water, which binds concurrently with the ET step. The analyzed IR-transients disfavor carboxylic-acid deprotonation in the pre-ET step. Temperature-dependent amplitudes suggest thermal equilibria that determine how strongly the proton-removal step is reflected in the IR-transients. Unexpectedly, the proton-removal step is only weakly reflected in the 1400 cm-1 transients of PSII core complexes of a thermophilic cyanobacterium (T. elongatus).

Glucose Biosensor Based on Disposable Activated Carbon Electrodes Modified with Platinum Nanoparticles Electrodeposited on Poly(Azure A).

Herein, a novel electrochemical glucose biosensor based on glucose oxidase (GOx) immobilized on a surface containing platinum nanoparticles (PtNPs) electrodeposited on poly(Azure A) (PAA) previously electropolymerized on activated screen-printed carbon electrodes (GOx-PtNPs-PAA-aSPCEs) is reported. The resulting electrochemical biosensor was validated towards glucose oxidation in real samples and further electrochemical measurement associated with the generated H2O2. The electrochemical biosensor showed an excellent sensitivity (42.7 µA mM-1 cm-2), limit of detection (7.6 µM), linear range (20 µM-2.3 mM), and good selectivity towards glucose determination. Furthermore, and most importantly, the detection of glucose was performed at a low potential (0.2 V vs. Ag). The high performance of the electrochemical biosensor was explained through surface exploration using field emission SEM, XPS, and impedance measurements. The electrochemical biosensor was successfully applied to glucose quantification in several real samples (commercial juices and a plant cell culture medium), exhibiting a high accuracy when compared with a classical spectrophotometric method. This electrochemical biosensor can be easily prepared and opens up a good alternative in the development of new sensitive glucose sensors.

Evaluating Face2Gene as a Tool to Identify Cornelia de Lange Syndrome by Facial Phenotypes.

Characteristic or classic phenotype of Cornelia de Lange syndrome (CdLS) is associated with a recognisable facial pattern. However, the heterogeneity in causal genes and the presence of overlapping syndromes have made it increasingly difficult to diagnose only by clinical features. DeepGestalt technology, and its app Face2Gene, is having a growing impact on the diagnosis and management of genetic diseases by analysing the features of affected individuals. Here, we performed a phenotypic study on a cohort of 49 individuals harbouring causative variants in known CdLS genes in order to evaluate Face2Gene utility and sensitivity in the clinical diagnosis of CdLS. Based on the profile images of patients, a diagnosis of CdLS was within the top five predicted syndromes for 97.9% of our cases and even listed as first prediction for 83.7%. The age of patients did not seem to affect the prediction accuracy, whereas our results indicate a correlation between the clinical score and affected genes. Furthermore, each gene presents a different pattern recognition that may be used to develop new neural networks with the goal of separating different genetic subtypes in CdLS. Overall, we conclude that computer-assisted image analysis based on deep learning could support the clinical diagnosis of CdLS.

ISG15 governs mitochondrial function in macrophages following vaccinia virus infection.

The interferon (IFN)-stimulated gene 15 (ISG15) encodes one of the most abundant proteins induced by interferon, and its expression is associated with antiviral immunity. To identify protein components implicated in IFN and ISG15 signaling, we compared the proteomes of ISG15-/- and ISG15+/+ bone marrow derived macrophages (BMDM) after vaccinia virus (VACV) infection. The results of this analysis revealed that mitochondrial dysfunction and oxidative phosphorylation (OXPHOS) were pathways altered in ISG15-/- BMDM treated with IFN. Mitochondrial respiration, Adenosine triphosphate (ATP) and reactive oxygen species (ROS) production was higher in ISG15+/+ BMDM than in ISG15-/- BMDM following IFN treatment, indicating the involvement of ISG15-dependent mechanisms. An additional consequence of ISG15 depletion was a significant change in macrophage polarization. Although infected ISG15-/- macrophages showed a robust proinflammatory cytokine expression pattern typical of an M1 phenotype, a clear blockade of nitric oxide (NO) production and arginase-1 activation was detected. Accordingly, following IFN treatment, NO release was higher in ISG15+/+ macrophages than in ISG15-/- macrophages concomitant with a decrease in viral titer. Thus, ISG15-/- macrophages were permissive for VACV replication following IFN treatment. In conclusion, our results demonstrate that ISG15 governs the dynamic functionality of mitochondria, specifically, OXPHOS and mitophagy, broadening its physiological role as an antiviral agent.

More Than One HMG-CoA Lyase: The Classical Mitochondrial Enzyme Plus the Peroxisomal and the Cytosolic Ones.

There are three human enzymes with HMG-CoA lyase activity that are able to synthesize ketone bodies in different subcellular compartments. The mitochondrial HMG-CoA lyase was the first to be described, and catalyzes the cleavage of 3-hydroxy-3-methylglutaryl CoA to acetoacetate and acetyl-CoA, the common final step in ketogenesis and leucine catabolism. This protein is mainly expressed in the liver and its function is metabolic, since it produces ketone bodies as energetic fuels when glucose levels are low. Another isoform is encoded by the same gene for the mitochondrial HMG-CoA lyase (HMGCL), but it is located in peroxisomes. The last HMG-CoA lyase to be described is encoded by a different gene, HMGCLL1, and is located in the cytosolic side of the endoplasmic reticulum membrane. Some activity assays and tissue distribution of this enzyme have shown the brain and lung as key tissues for studying its function. Although the roles of the peroxisomal and cytosolic HMG-CoA lyases remain unknown, recent studies highlight the role of ketone bodies in metabolic remodeling, homeostasis, and signaling, providing new insights into the molecular and cellular function of these enzymes.