Activation Of Mitochondrial Respiration Research Articles

Isomerization and phosphorylation significantly influence the effect of beta amyloid on blood‐brain barrier endothelial cells

AbstractBackgroundThe blood‐brain barrier (BBB) is typically compromised in Alzheimer’s disease (AD): its permeability and intracellular parameters of the endothelial cells are affected. AD‐related amyloid isoforms –Aβ42, isoD7‐Aβ42 and pS8‐Aβ42, inducing amyloidogenesis in a different mode – may have peripheral origin and affect the BBB cells. This study aimed to assess this effect.MethodMouse brain endothelial cells bEnd.3 underwent incubation with 10 µM of Aβ42, isoD7‐Aβ42 and pS8‐Aβ42 for 4, 24 and 48 hours to assess nitric oxide (NO), glutathione (GSH), reactive oxygen species (ROS), mitochondrial potential, Ca2+, cell viability by flow cytometry and mitochondrial functioning by Seahorse technology. To analyze the integrity of the cellular monolayer, the expression level of tight junction proteins and permeability of bEnd.3 monolayer to fluorescent tracers were assessed by Western Blotting and transwell‐modeling, correspondingly .ResultpS8‐Aβ42 and isoD7‐Aβ42 effects on the redox status of the cells and mitochondria functioning differ from the effects of Aβ42. Even at 4 hours, Aβ42 and isoD7‐Aβ42 induce immense changes in mitochondrial potential and NO level but pS8‐Aβ42 does not. Prolonged incubation with three isoforms leads to an increase in ROS, and greater increase is observed after incubation with isoD7‐Aβ42. pS8‐Aβ42 induces ROS increase later than other isoforms. Above all, incubation with pS8‐Aβ42 results in a less pronounced NO и GSH growth compared with Aβ42 и isoD7‐Aβ42 which induce activation of mitochondrial respiration and increase in mitochondrial potential, compared with pS8‐Aβ42. IsoD7‐Aβ42 is mostly toxic to bEnd.3 cells. Although Aβ42 and isoD7‐Aβ42 significantly affect redox parameters of bEnd.3 cells, their effects on BBB permeability and tight junction protein expression are less pronounced. Our data demonstrate that inhibition of NO synthases and NMDA receptors induces changes in BBB cells’ response to Aβ peptides.ConclusionAmyloid isoforms differently affect redox parameters of BBB cells significantly modulating mitochondria functioning. Iso‐Aβ42 induces higher cytotoxicity, thus, being a more pathogenic form. Post‐translational modifications of Aβ42 alter the amyloid effect on the BBB cells. It may result in different changes in BBB functioning and its permeability under amyloid isoforms.This research was funded by Russian Science Foundation (Grant No. #19‐74‐30007).

Cardioprotective effect of Interleukin-11 against warm ischemia-reperfusion injury in a rat heart donor model

Shortage of donor organs for heart transplantation is a worldwide problem. Donation after circulatory death (DCD) has been proposed to expand the donor pool. However, in contrast to the donation after brain death that undergoes immediate cold preservation, warm ischemia and subsequent reperfusion injury are inevitable in DCD. It has been reported that interleukin-11 (IL-11) mitigates ischemia-reperfusion injury in rodent models of myocardial infarction and donation after brain death heart transplantation. We hypothesized that IL-11 also offers benefit to warm ischemia in an experimental model of cardiac transplantation that resembles DCD. The hearts of naïve male Sprague Dawley rats (n = 15/group) were procured, subjected to 25-min warm ischemia, and reperfused for 60 min using Langendorff apparatus. IL-11 or saline was administered intravenously before the procurement, added to maintenance buffer, and infused via perfusion during reperfusion. IL-11 group exhibited significantly better cardiac function post-reperfusion. Severely damaged mitochondria was found in the electron microscopic analysis of control hearts whereas the mitochondrial structure was better preserved in the IL-11 treated hearts. Immunoblot analysis using neonatal rat cardiomyocytes revealed increased signal transducer and activator of transcription 3 (STAT3) phosphorylation at Ser727 after IL-11 treatment, suggesting its role in mitochondrial protection. Consistent with expected activation of mitochondrial respiration by mitochondrial STAT3, immunohistochemical staining demonstrated a higher mitochondrial cytochrome c oxidase subunit 2 expression. In summary, IL-11 protects the heart from warm ischemia reperfusion injury by alleviating mitochondrial injury and could be a viable therapeutic option for DCD heart transplantation.

Comparing Mitochondrial Activity, Oxidative Stress Tolerance, and Longevity of Thirteen Ascomycota Yeast Species.

Aging is characterized by a number of hallmarks including loss of mitochondrial homeostasis and decay in stress tolerance, among others. Unicellular eukaryotes have been widely used to study chronological aging. As a general trait, calorie restriction and activation of mitochondrial respiration has been proposed to contribute to an elongated lifespan. Most aging-related studies have been conducted with the Crabtree-positive yeasts Saccharomyces cerevisiae and Schizosaccharomyces pombe, and with deletion collections deriving from these conventional yeast models. We have performed an unbiased characterization of longevity using thirteen fungi species, including S. cerevisiae and S. pombe, covering a wide range of the Ascomycota clade. We have determined their mitochondrial activity by oxygen consumption, complex IV activity, and mitochondrial redox potential, and the results derived from these three methodologies are highly overlapping. We have phenotypically compared the lifespans of the thirteen species and their capacity to tolerate oxidative stress. Longevity and elevated tolerance to hydrogen peroxide are correlated in some but not all yeasts. Mitochondrial activity per se cannot anticipate the length of the lifespan. We have classified the strains in four groups, with members of group 1 (Kluyveromyces lactis, Saccharomyces bayanus and Lodderomyces elongisporus) displaying high mitochondrial activity, elevated resistance to oxidative stress, and elongated lifespan.

1593-P: Insulin-Degrading Enzyme (IDE) Regulates Glucagon Signalling and Mitochondrial Respiration in Hepatocytes

IDE is a ubiquitous metalloprotease with cytosolic and mitochondrial subcellular localization that degrades insulin and glucagon. People with T2D and diet-induced obese mice show lower hepatic IDE levels. We revealed a key role of IDE on the insulin-mediated repression of hepatic gluconeogenesis, but its function on glucagon-dependent activation of gluconeogenesis and mitochondrial respiration in hepatocytes remains completely unknown. Here, we aim to elucidate the role of IDE on glucagon signalling and its impact on gluconeogenesis and energy metabolism in hepatocytes. Liver homogenized and primary hepatocytes obtained from L-IDE-KO mice (deletion of IDE in liver) showed decreased expression of glucagon receptor (~60%), CREB protein (~40%), and lower phosphorylation of CREB (~50%) upon glucagon stimulation compared to controls. Similar results were found in AML12-shRNA-Ide cells, in which IDE protein levels were reduced by ~50%. Additionally, glucagon stimulation resulted in lower (~30%) cAMP levels and diminished phosphorylation of PKA substrates in AML12-shRNA-Ide. Surprisingly, these alterations in glucagon signalling paralleled with ~20-fold increases in the expression of the gluconeogenic genes G6p6 and Pck1. Of note, basal and uncoupler-stimulated respiration increased ~4-fold in AML12-shRNA-Ide in parallel with a ~2-fold increment of mitochondrial and glycolytic ATP production. Finally, similar mitochondrial phenotype was found in human hepatocytes lacking IDE (HepG2-IDE-KO cells), which exhibited higher FoxO1 levels and fragmented mitochondria. The effects on mitochondrial respiration were independent of IDE's proteolytic activity. In summary, reduced IDE expression in hepatocytes has a deleterious effect on glucagon signalling leading to up-regulated gluconeogenesis and mitochondrial respiration. We conclude that IDE is a mechanistic link to couple hepatic gluconeogenesis with mitochondrial energy production. Disclosure C.M. González-Casimiro: None. P. Cámara-Torres: None. B. Merino: None. J. Santo-Domingo: None. M.A. de la Fuente: None. A. Alonso: None. I. Cozar-Castellano: None. G. Perdomo: None. Funding Ministerio de Ciencia e Innovación-Spain (PID2019-110496RB-C22)

A Window to the Potassium World. The Evidence of Potassium Energetics in the Mitochondria and Identity of the Mitochondrial ATP-Dependent K+ Channel.

The conclusions made in the three papers published in Function by Juhaszova et al. [Function, 3, 2022, zqab065, zqac001, zqac018], can be seen as a breakthrough in bioenergetics and mitochondrial medicine. For more than half a century, it has been believed that mitochondrial energetics is solely protonic and is based on the generation of electrochemical potential of hydrogen ions across the inner mitochondrial membrane upon oxidation of respiratory substrates, resulting in the generation of ATP via reverse transport of protons through the ATP synthase complex. Juhaszova et al. demonstrated that ATP synthase transfers not only protons, but also potassium ions, with the generation of ATP. This mechanism seems logical, given the fact that in eukaryotic cells, the concentration of potassium ions is several million times higher than the concentration of protons. The transport of K+ through the ATP synthase was enhanced by the activators of mitochondrial ATP-dependent K+ channel (mK/ATP), leading to the conclusion that ATP synthase is the material essence of mK/ATP. Beside ATP generation, the transport of osmotically active K+ to the mitochondrial matrix is accompanied by water entry to the matrix, leading to an increase in the matrix volume and activation of mitochondrial respiration with the corresponding increase in the ATP synthesis, which suggests an advantage of such transport for energy production. The driving force for K+ transport into the mitochondria is the membrane potential; an excess of K+ is exported from the matrix by the hypothetical K+/H+ exchangers. Inhibitory factor 1 (IF1) plays an important role in the activation of mK/ATP by increasing the chemo-mechanical efficiency of ATP synthase, which may be a positive factor in the protective anti-ischemic signaling.

Deletion of GPR30 Drives the Activation of Mitochondrial Uncoupling Respiration to Induce Adipose Thermogenesis in Female Mice.

Thermogenic adipocytes possess a promising approach to combat obesity with its capability promoting energy metabolism. We previously discovered that deletion of GPR30 (GPRKO), a presumably membrane-associated estrogen receptor, protected female mice from developing obesity, glucose intolerance, and insulin resistance when challenged with a high-fat diet (HFD). In vivo, the metabolic phenotype of wild type (WT) and GPRKO female mice were measured weekly. Acute cold tolerance test was performed. Ex vivo, mitochondrial respiration of brown adipose tissue (BAT) was analyzed from diet-induced obese female mice of both genotypes. In vitro, stromal vascular fractions (SVF) were isolated for beige adipocyte differentiation to investigate the role of GPR30 in thermogenic adipocyte. Deletion of GPR30 protects female mice from hypothermia and the mitochondria in BAT are highly energetic in GPRKO animals while the WT mitochondria remain in a relatively quiescent stage. Consistently, GPR30 deficiency enhances beige adipocyte differentiation in white adipose tissue (WAT) and activates the thermogenic browning of subcutaneous WAT due to up-regulation of UCP-1, which thereby protects female mice from HFD-induced obesity. GPR30 is a negative regulator of thermogenesis, which at least partially contributes to the reduced adiposity in the GPRKO female mice. Our findings provide insight into the mechanism by which GPR30 regulates fat metabolism and adiposity in female mice exposed to excess calories, which may be instrumental in the development of new therapeutic strategies for obesity.

Exogenous mitochondrial transfer and endogenous mitochondrial fission facilitate AML resistance to OxPhos inhibition

Acute myeloid leukemia (AML) cells are highly dependent on oxidative phosphorylation (OxPhos) for survival, and they continually adapt to fluctuations in nutrient and oxygen availability in the bone marrow (BM) microenvironment. We investigated how the BM microenvironment affects the response to OxPhos inhibition in AML by using a novel complex I OxPhos inhibitor, IACS-010759. Cellular adhesion, growth, and apoptosis assays, along with measurements of expression of mitochondrial DNA and generation of mitochondrial reactive oxygen species indicated that direct interactions with BM stromal cells triggered compensatory activation of mitochondrial respiration and resistance to OxPhos inhibition in AML cells. Mechanistically, inhibition of OxPhos induced transfer of mitochondria derived from mesenchymal stem cells (MSCs) to AML cells via tunneling nanotubes under direct-contact coculture conditions. Inhibition of OxPhos also induced mitochondrial fission and increased functional mitochondria and mitophagy in AML cells. Mitochondrial fission is known to enhance cell migration, so we used electron microscopy to observe mitochondrial transport to the leading edge of protrusions of AML cells migrating toward MSCs. We further demonstrated that cytarabine, a commonly used antileukemia agent, increased mitochondrial transfer of MSCs to AML cells triggered by OxPhos inhibition. Our findings indicate an important role of exogenous mitochondrial trafficking from BM stromal cells to AML cells as well as endogenous mitochondrial fission and mitophagy in the compensatory adaptation of leukemia cells to energetic stress in the BM microenvironment.

AML-108: Exogenous Mitochondrial Transfer and Endogenous Mitochondrial Fission Facilitate Resistance to OxPhos Inhibition in AML

<h3>Context:</h3> Oxidative phosphorylation (OxPhos) inhibition induces mitochondrial trafficking from BM stromal cells to AML and is accompanied by mitochondrial fission and mitophagy. The mitochondrial coupling and crosstalk with BM stromal cells facilitate the development of resistance to OxPhos inhibition in AML cells. <h3>Objective:</h3> Acute myeloid leukemia (AML) cells are highly dependent on OxPhos for survival and continually adapt to fluctuations in nutrient and oxygen availability in the environment. We investigated how the BM microenvironment affects the response to OxPhos inhibition in AML by using a novel complex I OxPhos inhibitor, IACS-010759. <h3>Design:</h3> Primary AML cells from 26 patients, primary normal MSCs, and AML cell lines were utilized and treated with the OxPhos inhibitor IACS-0107598 and/or cytarabine. Cap analysis of gene expression transcriptome analyses for AML PDXs, primary samples, and AML cell lines has been performed. We performed an extracellular flux assay to measure the oxygen consumption rate. To visualize mitochondria, AML cell lines and MSCs were stably transfected with mitochondria-targeted PDHA1-GFP and -dsRed, respectively. Electron microscopy and immunoelectron microscopy analysis have been performed. <h3>Results:</h3> Cellular adhesion, growth, and apoptosis assays, along with measurements of mtDNA expression and mitochondrial reactive oxygen species generation, indicated that direct interactions with BM stromal cells triggered the compensatory activation of mitochondrial respiration and resistance to OxPhos inhibition in AML cells. Mechanistically, OxPhos inhibition induced (1) transfer of mesenchymal stem cell (MSC)-derived mitochondria to AML cells via tunneling nanotubes under direct-contact co-culture conditions and (2) mitochondrial fission with an increase in functional mitochondria and mitophagy in AML cells. Mitochondrial fission is known to enhance cell migration, and we observed mitochondrial transport to the leading edge of protrusions of migrating AML cells toward MSCs by electron microscopy analysis. We further demonstrated that cytarabine, a commonly used antileukemia agent, increased IACS-010759-triggered mitochondrial transfer from MSCs to AML cells. <h3>Conclusions:</h3> Our findings indicate an important role of exogenous mitochondrial trafficking from BM stromal cells to AML cells and of endogenous mitochondrial fission and mitophagy in the compensatory adaptation of leukemia cells to energetic stress in the BM microenvironment.

Interleukin-33 regulates metabolic reprogramming of the retinal pigment epithelium in response to immune stressors.

It remains unresolved how retinal pigment epithelial cell metabolism is regulated following immune activation to maintain retinal homeostasis and retinal function. We exposed retinal pigment epithelium (RPE) to several stress signals, particularly Toll-like receptor stimulation, and uncovered an ability of RPE to adapt their metabolic preference on aerobic glycolysis or oxidative glucose metabolism in response to different immune stimuli. We have identified interleukin-33 (IL-33) as a key metabolic checkpoint that antagonizes the Warburg effect to ensure the functional stability of the RPE. The identification of IL-33 as a key regulator of mitochondrial metabolism suggests roles for the cytokine that go beyond its extracellular “alarmin” activities. IL-33 exerts control over mitochondrial respiration in RPE by facilitating oxidative pyruvate catabolism. We have also revealed that in the absence of IL-33, mitochondrial function declined and resultant bioenergetic switching was aligned with altered mitochondrial morphology. Our data not only shed new light on the molecular pathway of activation of mitochondrial respiration in RPE in response to immune stressors but also uncover a potentially novel role of nuclear intrinsic IL-33 as a metabolic checkpoint regulator.

Singlet oxygen stimulates mitochondrial bioenergetics in brain cells

Oxygen, in form of reactive oxygen species (ROS), has been shown to participate in oxidative stress, one of the major triggers for pathology, but also is a main contributor to physiological processes. Recently, it was found that 1267 nm irradiation can produce singlet oxygen without photosensitizers. We used this phenomenon to study the effect of laser-generated singlet oxygen on one of the major oxygen-dependent processes, mitochondrial energy metabolism. We have found that laser-induced generation of 1O2 in neurons and astrocytes led to the increase of mitochondrial membrane potential, activation of NADH- and FADH-dependent respiration, and importantly, increased the rate of maximal respiration in isolated mitochondria. The activation of mitochondrial respiration stimulated production of ATP in these cells. Thus, we found that the singlet oxygen generated by 1267 nm laser pulse works as an activator of mitochondrial respiration and ATP production in the brain.

Cbl Negatively Regulates NLRP3 Inflammasome Activation through GLUT1-Dependent Glycolysis Inhibition.

Activation of the nod-like receptor 3 (NLRP3) inflammasomes is crucial for immune defense, but improper and excessive activation causes inflammatory diseases. We previously reported that Cbl plays a pivotal role in suppressing NLRP3 inflammasome activation by inhibiting Pyk2-mediated apoptosis-associated speck-like protein containing a CARD (ASC) oligomerization. Here, we showed that Cbl dampened NLRP3 inflammasome activation by inhibiting glycolysis, as demonstrated with Cbl knockout cells and treatment with the Cbl inhibitor hydrocotarnine. We revealed that the inhibition of Cbl promoted caspase-1 cleavage and interleukin (IL)-1β secretion through a glycolysis-dependent mechanism. Inhibiting Cbl increased cellular glucose uptake, glycolytic capacity, and mitochondrial oxidative phosphorylation capacity. Upon NLRP3 inflammasome activation, inhibiting Cbl increased glycolysis-dependent activation of mitochondrial respiration and increased the production of reactive oxygen species, which contributes to NLRP3 inflammasome activation and IL-1β secretion. Mechanistically, inhibiting Cbl increased surface expression of glucose transporter 1 (GLUT1) protein through post-transcriptional regulation, which increased cellular glucose uptake and consequently raised glycolytic capacity, and in turn enhanced NLRP3 inflammasome activation. Together, our findings provide new insights into the role of Cbl in NLRP3 inflammasome regulation through GLUT1 downregulation. We also show that a novel Cbl inhibitor, hydrocortanine, increased NLRP3 inflammasome activity via its effect on glycolysis.

Redox-mediated kick-start of mitochondrial energy metabolism drives resource-efficient seed germination

Seeds preserve a far developed plant embryo in a quiescent state. Seed metabolism relies on stored resources and is reactivated to drive germination when the external conditions are favorable. Since the switchover from quiescence to reactivation provides a remarkable case of a cell physiological transition we investigated the earliest events in energy and redox metabolism of Arabidopsis seeds at imbibition. By developing fluorescent protein biosensing in intact seeds, we observed ATP accumulation and oxygen uptake within minutes, indicating rapid activation of mitochondrial respiration, which coincided with a sharp transition from an oxidizing to a more reducing thiol redox environment in the mitochondrial matrix. To identify individual operational protein thiol switches, we captured the fast release of metabolic quiescence in organello and devised quantitative iodoacetyl tandem mass tag (iodoTMT)-based thiol redox proteomics. The redox state across all Cys peptides was shifted toward reduction from 27.1% down to 13.0% oxidized thiol. A large number of Cys peptides (412) were redox switched, representing central pathways of mitochondrial energy metabolism, including the respiratory chain and each enzymatic step of the tricarboxylic acid (TCA) cycle. Active site Cys peptides of glutathione reductase 2, NADPH-thioredoxin reductase a/b, and thioredoxin-o1 showed the strongest responses. Germination of seeds lacking those redox proteins was associated with markedly enhanced respiration and deregulated TCA cycle dynamics suggesting decreased resource efficiency of energy metabolism. Germination in aged seeds was strongly impaired. We identify a global operation of thiol redox switches that is required for optimal usage of energy stores by the mitochondria to drive efficient germination.

Inhibition of receptor-interacting protein kinase 1 improves experimental non-alcoholic fatty liver disease

In non-alcoholic fatty liver disease (NAFLD), hepatocytes can undergo necroptosis: a regulated form of necrotic cell death mediated by the receptor-interacting protein kinase (RIPK) 1. Herein, we assessed the potential for RIPK1 and its downstream effector mixed lineage kinase domain-like protein (MLKL) to act as therapeutic targets and markers of activity in NAFLD. C57/BL6J-mice were fed a normal chow diet or a high-fat diet (HFD). The effect of RIPA-56, a highly specific inhibitor of RIPK1, was evaluated in HFD-fed mice and in primary human steatotic hepatocytes. RIPK1 and MLKL concentrations were measured in the serum of patients with NAFLD. When used as either a prophylactic or curative treatment for HFD-fed mice, RIPA-56 caused a downregulation of MLKL and a reduction of liver injury, inflammation and fibrosis, characteristic of non-alcoholic steatohepatitis (NASH), as well as of steatosis. This latter effect was reproduced by treating primary human steatotic hepatocytes with RIPA-56 or necrosulfonamide, a specific inhibitor of human MLKL, and by knockout (KO) of Mlklin fat-loaded AML-12 mouse hepatocytes. Mlkl-KO led to activation of mitochondrial respiration and an increase in β-oxidation in steatotic hepatocytes. Along with decreased MLKL activation, Ripk3-KO mice exhibited increased activities of the liver mitochondrial respiratory chain complexes in experimental NASH. In patients with NAFLD, serum concentrations of RIPK1 and MLKL increased in correlation with activity. The inhibition of RIPK1 improves NASH features in HFD-fed mice and reverses steatosis via an MLKL-dependent mechanism that, at least partly, involves an increase in mitochondrial respiration. RIPK1 and MLKL are potential serum markers of activity and promising therapeutic targets in NAFLD. There are currently no pharmacological treatment options for non-alcoholic fatty liver disease (NAFLD), which is now the most frequent liver disease. Necroptosis is a regulated process of cell death that can occur in hepatocytes during NAFLD. Herein, we show that RIPK1, a gatekeeper of the necroptosis pathway that is activated in NAFLD, can be inhibited by RIPA-56 to reduce not only liver injury, inflammation and fibrosis, but also steatosis in experimental models. These results highlight the potential of RIPK1 as a therapeutic target in NAFLD.

Oxphos Inhibition Induces Formation of Tunneling Nanotubes in AML Cells and Facilitates Mitochondrial Transfer from BM Stroma to AML That Contributes to Microenvironment-Mediated Drug-Resistance of AML

Oxphos Inhibition Induces Formation of Tunneling Nanotubes in AML Cells and Facilitates Mitochondrial Transfer from BM Stroma to AML That Contributes to Microenvironment-Mediated Drug-Resistance of AML

Implication of Mitochondrial NO/cGMP/PKG Signaling System in the Activation and Inhibition of Mitochondrial Respiration by L-Arginine and NO Donors

The involvement of the mitochondrial calcium-dependent NO synthase (mtNOS) in the regulation of mitochondrial respiration has not been sufficiently studied. Moreover, the possible functioning of a mitochondrial signaling system involving mtNOS/guanylate cyclase (GC)/protein kinase G (PKG) and the impact of this system (mtNOS/GC/PKG-SS) on mitochondrial respiration have not yet been analyzed. To investigate this issue, we performed experiments on isolated rat liver using specific inhibitors of NOS, GC, and PKG. The high rate of mitochondrial respiration was supported by pyruvate and glutamate or by succinate in the presence of hexokinase, glucose, and ADP. It was shown that L-arginine and the NO donor sodium nitroprusside (SNP) exert concentration-dependent effects on the mitochondrial respiration rate. At low concentrations, L‑arginine (up to 200 μM) and SNP (up to100 μM) activated mitochondrial respiration. The inhibitors of NOS, GC, and PKG eliminated this effect indicating that mtNOS/GC/PKG-SS is involved in the activation of respiration. At high concentrations, L-arginine and SNP, on the contrary, inhibited respiration. Under these conditions, the inhibitors of GC and PKG enhanced the inhibition of respiration, which indicates an opposite effect of the excess of NO and PKG on the mitochondrial respiration. The results suggest that the functioning of calcium-dependent mtNOS/GC/PKG-SS can ensure the activation of respiration at low concentrations of L-arginine or SNP in the medium.

Chitosan-induced enhanced expression and activation of alternative oxidase confer tolerance to salt stress in maize seedlings

This study aimed to investigate the possible alleviating effect of chitosan on salt-induced growth retardation and oxidative stress and to elucidate whether this effect is linked to activation of mitochondrial respiration on the basis of alternative respiration in maize seedlings. Salt stress significantly reduced root length and plant height in comparison to the control, whereas foliar application of chitosan ameliorated the adverse effect of salinity to a certain degree. Moreover, chitosan resulted in plant growth promotion as compared to unstressed seedlings. The separate applications of chitosan and salt had a stimulatory effect on the activities of antioxidant enzymes; however, combined application of chitosan and salt were more effective than that of chitosan or salt alone. Similarly, mitochondrial total respiration rate (Vt) and alternative respiration capacity (Valt) were increased by separate applications of chitosan and salt; however, the combination of chitosan and salt gave the highest values for these parameters. The highest values of Valt/Vt was recorded at seedlings treated with salt plus chitosan. Similarly, cytochrome respiration capacity was also increased by chitosan in both stress-free and stressed conditions. In addition, AOX1, encoding alternative oxidase, was significantly upregulated by chitosan and/or salt. The maximum transcript level was recorded at seedlings treated with salt plus chitosan. Chitosan also significantly decreased superoxide anion and hydrogen peroxide contents and lipid peroxidation level under normal and the stressed conditions. These results suggest that the mitigating effect of chitosan on salt stress is linked to activation of alternative respiration at biochemical and molecular level.

Cardiolipin-Dependent Mitophagy Guides Outcome after Traumatic Brain Injury.

Mitochondrial energy production is essential for normal brain function. Traumatic brain injury (TBI) increases brain energy demands, results in the activation of mitochondrial respiration, associated with enhanced generation of reactive oxygen species. This chain of events triggers neuronal apoptosis via oxidation of a mitochondria-specific phospholipid, cardiolipin (CL). One pathway through which cells can avoid apoptosis is via elimination of damaged mitochondria by mitophagy. Previously, we showed that externalization of CL to the mitochondrial surface acts as an elimination signal in cells. Whether CL-mediated mitophagy occurs in vivo or its significance in the disease processes are not known. In this study, we showed that TBI leads to increased mitophagy in the human brain, which was also detected using TBI models in male rats. Knockdown of CL synthase, responsible for de novo synthesis of CL, or phospholipid scramblase-3, responsible for CL translocation to the outer mitochondrial membrane, significantly decreased TBI-induced mitophagy. Inhibition of mitochondrial clearance by 3-methyladenine, mdivi-1, or phospholipid scramblase-3 knockdown after TBI led to a worse outcome, suggesting that mitophagy is beneficial. Together, our findings indicate that TBI-induced mitophagy is an endogenous neuroprotective process that is directed by CL, which marks damaged mitochondria for elimination, thereby limiting neuronal death and behavioral deficits.SIGNIFICANCE STATEMENT Traumatic brain injury (TBI) increases energy demands leading to activation of mitochondrial respiration associated with enhanced generation of reactive oxygen species and resultant damage to mitochondria. We demonstrate that the complete elimination of irreparably damaged organelles via mitophagy is activated as an early response to TBI. This response includes translocation of mitochondria phospholipid cardiolipin from the inner membrane to the outer membrane where externalized cardiolipin mediates targeted protein light chain 3-mediated autophagy of damaged mitochondria. Our data on targeting phospholipid scramblase and cardiolipin synthase in genetically manipulated cells and animals strongly support the essential role of cardiolipin externalization mechanisms in the endogenous reparative plasticity of injured brain cells. Furthermore, successful execution and completion of mitophagy is beneficial in the context of preservation of cognitive functions after TBI.

Increased mitochondrial respiration promotes survival from endoplasmic reticulum stress.

Protein misfolding in the endoplasmic reticulum (ER) is accompanied by adaptive cellular responses to promote cell survival. We now show that activation of mitochondrial respiration is a critical component of an adaptive ER stress response, requiring the unfolded protein response (UPR) sensor Ire1, and also calcium signaling via calcineurin. In yeast and mammalian cells lacking Ire1 or calcineurin, respiratory activation is impaired in response to ER stress; accumulation of mitochondrial reactive oxygen species (ROS) triggers cell death as abrogation of ROS by antioxidants or loss of the electron transport chain (in yeast) can rescue cells from death. Significantly, cells are rescued from ER stress-induced death by mitochondrial uncoupling by CCCP to increase O2 consumption (and increase the efficiency of electron transfer). Remarkably, genetic and pharmacologic strategies to promote mitochondrial biogenesis and increase O2 consumption also alleviate ER stress-mediated ROS and death in yeast and mammalian cells. Moreover, in a yeast genetic screen, three mitochondrial proteins Mrx9, Mrm1, and Aim19 that increase mitochondrial biogenesis were identified as high copy suppressors of ER stress-mediated cell death. Our results show that enhanced mitochondrial biogenesis, linked to improved efficiency of the electron transport chain, is a powerful strategy to block ROS accumulation and promote cell survival during ER stress in eukaryotic cells.

Fundamental role of warburg effect in various pathophysiological processes

In this review article, the role of Warburg effect in various pathologies is discussed using septic shock as the base model. I had proposed a slightly extended Warburg effect which I would like to call it as “Warburg common pathogenesis model or Warburg differentiation–dedifferentiation effect,” which has the potential to explain septic shock and sepsis-associated multiorgan dysfunction and many other major pathologies such as systemic hypertension, pulmonary arterial hypertension, congestive heart failure, diabetes mellitus, asthma, acute respiratory distress syndrome, and acute kidney injury, implying that most of the diseases may have a common pathogenesis as the underlying mechanism. Increased nitric oxide (NO) in sepsis via inducible nitric oxide synthase (iNOS) or any respiratory poison, in general, irreversibly inhibits the mitochondrial respiration and shifts the metabolic phenotype of the cell from oxidative phosphorylation to glycolytic phenotype, and the change in metabolic phenotype is followed by the change in cell phenotype from the normal adult dynamic differentiation state to irreversible dedifferentiation states – embryonic phenotype, synthetic/proliferative phenotype, and cancer phenotype. This dedifferentiated state switching can be seen as the cells' local survival strategy in response to injuries, but returning to their primitive forms leads to disorder and ends in global collapse of the organ systems and organism which requires order in terms of differentiation. Treatment in most of the pathologies should aim at reversing Warburg effect by the activation of mitochondrial respiration, thereby decreasing the aerobic glycolysis and changing the cell to its normal adult dynamic differentiation phenotype, i.e. all the drugs are used here as differentiation therapy. Adrenergic blockers and ascorbic acid may be the main treatment options, which are already used by some research groups. Even though high NO via iNOS was involved in most of the pathologies including sepsis, any substance that inhibits or uncouples the mitochondrial respiration can initiate this Warburg effect and irreversible dedifferentiation. A mild and reversible Warburg differentiation–dedifferentiation effect may be necessary for normal functioning, and the same effect in exaggerated and irreversible way leading to irreversible dedifferentiation states may be the underlying mechanism in most of the diseases including septic shock.

Multilayered control of peroxisomal activity upon salt stress in Saccharomyces cerevisiae.

Peroxisomes are dynamic organelles and the sole location for fatty acid β-oxidation in yeast cells. Here, we report that peroxisomal function is crucial for the adaptation to salt stress, especially upon sugar limitation. Upon stress, multiple layers of control regulate the activity and the number of peroxisomes. Activated Hog1 MAP kinase triggers the induction of genes encoding enzymes for fatty acid activation, peroxisomal import and β-oxidation through the Adr1 transcriptional activator, which transiently associates with genes encoding fatty acid metabolic enzymes in a stress- and Hog1-dependent manner. Moreover, Na+ and Li+ stress increases the number of peroxisomes per cell in a Hog1-independent manner, which depends instead of the retrograde pathway and the dynamin related GTPases Dnm1 and Vps1. The strong activation of the Faa1 fatty acyl-CoA synthetase, which specifically localizes to lipid particles and peroxisomes, indicates that adaptation to salt stress requires the enhanced mobilization of fatty acids from internal lipid stores. Furthermore, the activation of mitochondrial respiration during stress depends on peroxisomes, mitochondrial acetyl-carnitine uptake is essential for salt resistance and the number of peroxisomes attached to the mitochondrial network increases during salt adaptation, which altogether indicates that stress-induced peroxisomal β-oxidation triggers enhanced respiration upon salt shock.