ETC Complexes Research Articles

Efficacy Evaluation of a Tetracyclic Triterpene Compound Targeting Multiple Apoptosis Proteins for the Development of a Multiple Myeloma Treatment

Introduction:Multiple myeloma (MM) is an incurable hematologic cancer that originates in plasma cells and occurs primarily in older patients over 60. This study screened for hit compounds for MM treatments targeting mitochondria and apoptosis proteins. Further, non-clinical testing, including molecular docking analysis, was performed. Methods: Based on our previous studies, candidate compounds targeting mitochondria were screened (www.medchemexpress.com). The killing effect of the compounds on MM cell lines (RPMI8226, IM9, and U266) was examined using a sulforhodamine B assay. Apoptosis mechanisms were investigated in vitro. Complex immunocompromised mice were established by injecting RPMI8226-RFP-FLuc cells through the tail vein. Efficacy of the KBB-N2 activity was assessed following intraperitoneal (15 or 30 mg/kg/day) and tail vein (7 mg/kg/day) infusions for three days a week at daily intervals. Toxicity, pharmacokinetics, and molecular docking analyses were performed according to the established protocols. Results: The hit compound, KBB-N2, a tetracyclic triterpene, was identified as an MM-targeted compound with cytotoxic effects. The primary mechanism of action of KBB-N2 in MM cells was inhibiting the activity of the mitochondrial electron transport (ETC) complex, resulting in the generation of excess intracellular reactive oxygen species (ROS), which led to tumor cell death by ROS-mediated apoptosis. In addition, exposure to KBB-N2 inhibited the activity of catalase, a powerful antioxidant enzyme in MM cells. Severe suppression of normal hematopoietic stem cell function was observed with lenalidomide administration at a concentration of 25 μM. However, this toxicity was not observed when KBB-N2 was administered at doses up to 60 μM. KBB-N2 activity involves PARP cleavage and caspase activation in the apoptotic pathway. Furthermore, KBB-N2 inhibited cell growth and exerted cytotoxicity. KBB-N2 induced apoptosis by activating the P53 apoptosis pathway in MM cells. In vivo, all untreated tumor-bearing mice showed rapid tumor growth and severe plasmacytomas, which led to death within seven weeks. Mice treated with KBB-N2 had significantly inhibited tumor growth and longer survival times. The molecular docking assay revealed the optimal docking conformation binding energy of KBB-N2 with docking scores (kcal/M) of -7.3 and -9.8 for ETC complexes I and III, respectively. Conclusions: The triterpene compound, KBB-N2, was identified as a hit compound and may act as a targeted agent for MM treatment. The antimyeloma activity of KBB-N2 involves interference with the ETC complex and leads to MM cell death.

The Role of Graphene Oxide in the Inhibition of Migration and Invasion of Cancer Cells by Destroying Actin Cytoskeleton and via Inhibiting the Activities of ETC Complexes

Abstract: Cancer is one of the most important threats to public health. Cancer is characterized by cell proliferation that has eluded central endogenous control mechanisms. Cervical cancer is the third most common cancer among women, followed by skin cancer and breast cancer, the first and second most common causes, respectively. In developing countries, cervical cancer is usually the most common cancer in women and may account for 25% of all female cancers. Over the years, the diagnosis and treatment of cervical cancer have made rapid progress, resulting in a decline in the mortality and morbidity of the disease. Unfortunately, although surgery and radiotherapy have effective treatment options for early cervical cancer, poor prognosis is still a challenge in the treatment of metastatic cervical cancer. Therefore, it is very important to reveal the mechanism of cervical cancer and explore new therapies against tumor invasiveness. At present, it is reported that nanomaterials are used in the detection and treatment of various types of malignant tumors due to their different targeting effects in many fields, such as imaging, immune detection, chemotherapy, radiotherapy and immunotherapy. The cytotoxicity and inhibitory effects of graphene oxide (GO) on tumor invasion and metastasis were studied in recent studies using the human cervical cancer Hela cell line, as well as the probable mechanisms and signaling pathways involved. Here we collect the last reports, with focus on the role of GO in the inhibition of migration and invasion of cancer cells with the goal of exposing possible potential pathways to provide new insights for specific treatment of cancer.

Unveiling the Role of the Proton Gateway, Uncoupling Proteins (UCPs), in Cancer Cachexia

Uncoupling proteins (UCPs) are identified as carriers of proton ions between the mitochondrial inner membrane and the mitochondrial matrix. ATP is mainly generated through oxidative phosphorylation in mitochondria. The proton gradient is generated across the inner mitochondrial membrane and the mitochondrial matrix, which facilitates a smooth transfer of electrons across ETC complexes. Until now, it was thought that the role of UCPs was to break the electron transport chain and thereby inhibit the synthesis of ATP. UCPs allow protons to pass from the inner mitochondrial membrane to the mitochondrial matrix and decrease the proton gradient across the membrane, which results in decreased ATP synthesis and increased production of heat by mitochondria. In recent years, the role of UCPs in other physiological processes has been deciphered. In this review, we first highlighted the different types of UCPs and their precise location across the body. Second, we summarized the role of UCPs in different diseases, mainly metabolic disorders such as obesity and diabetes, cardiovascular complications, cancer, wasting syndrome, neurodegenerative diseases, and kidney complications. Based on our findings, we conclude that UCPs play a major role in maintaining energy homeostasis, mitochondrial functions, ROS production, and apoptosis. Finally, our findings reveal that mitochondrial uncoupling by UCPs may treat many diseases, and extensive clinical studies are required to meet the unmet need of certain diseases.

Ucp2 regulates neonatal cardiomyocytes proliferation through modulation of their mitochondrial metabolism

A major block to mammalian cardiac regeneration is the limited capacity of adult cardiomyocytes (CMs) to proliferate in response to injury. CMs proliferation is linked to oxidative metabolism: at birth, neonatal CMs endure a metabolic switch, with high reliance on fatty acid oxidation. This leads to increased mtROS production that has been shown to mediate their cell-cycle arrest. Thus, studying mitochondrial activity in the neonate is critical to understand constrains on CMs proliferation, regardless of their age. In this context, we focus of Ucp2, a mitochondrial carrier that plays a key role in regulation of cell fuel preferences and mtROS production. Our goal is to investigate whether Ucp2 can regulate neonatal CMs proliferation through modulation of their oxidative metabolism. Using inducible and complete Ucp2 KOs mice models together with RNAseq, flow cytometry and Seahorse analysis, we have addressed the role of Ucp2 in regulation of neonatal CMs proliferation, mtROS levels, mitochondrial biogenesis and metabolic activity. Being expressed at low levels during development, Ucp2 is upregulated from P1 to P7. Its expression then decreases to become barely detectable in the adult, thus correlating with CMs proliferative period. We show that neonatal Ucp2-/- CMs display a reduced mitochondrial respiration (OCR) that is not compensated by glycolysis. This low OCR is accompanied by a strong decrease in mitochondrial mass, together with alterations of ETC complexes expression, and to an increased reliance on glutaminolysis. In addition to decreased ATP/ADP ratio, we show that this impaired metabolic activity leads to reduction of CMs proliferation, a phenotype that is mediated by p38MAPK signaling pathway. We are currently assessing the consequences of loss of Ucp2 on neonatal heart metabolites levels and mitochondrial network. We hypothesize that Ucp2, through the export of metabolites from mitochondria to cytosol, takes part in mitochondrial retrograde signaling leading to regulation of CMs maturation and proliferation.

The intra-mitochondrial O-GlcNAcylation system rapidly modulates OXPHOS function and ROS release in the heart

Protein O-GlcNAcylation is increasingly recognized as an important cellular regulatory mechanism, in multiple organs including the heart. However, the mechanisms leading to O-GlcNAcylation in mitochondria and the consequences on their function remain poorly understood. In this study, we use an in vitro reconstitution assay to characterize the intra-mitochondrial O-GlcNAc system without potential cytoplasmic confounding effects. We compare the O-GlcNAcylome of isolated cardiac mitochondria with that of mitochondria acutely exposed to NButGT, a specific inhibitor of glycoside hydrolase. Amongst the 409 O-GlcNAcylated mitochondrial proteins identified, 191 display increased O-GlcNAcylation in response to NButGT. This is associated with enhanced Complex I (CI) activity, increased maximal respiration in presence of pyruvate-malate, and a striking reduction of mitochondrial ROS release, which could be related to O-GlcNAcylation of specific subunits of ETC complexes (CI, CIII) and TCA cycle enzymes. In conclusion, our work underlines the existence of a dynamic mitochondrial O-GlcNAcylation system capable of rapidly modifying mitochondrial function.

Dimethylglycine sodium salt activates Nrf2/SIRT1/PGC1α leading to the recovery of muscle stem cell dysfunction in newborns with intrauterine growth restriction

The objectives of this study were focused on the mechanism of mitochondrial dysfunction in skeletal muscle stem cells (MuSCs) from intrauterine growth restriction (IUGR) newborn piglets, and the relief of dimethylglycine sodium salt (DMG-Na) on MuSCs mitochondrial dysfunction by Nrf2/SIRT1/PGC1α network. In this study, six newborn piglets with normal birth weight (NBW) and six IUGR newborn piglets were slaughtered immediately after birth to obtain longissimus dorsi muscle (LM) samples. MuSCs were collected and divided into three groups: MuSCs from NBW newborn piglets (N), MuSCs from IUGR newborn piglets (I), and MuSCs from IUGR newborn piglets with 32 μmol DMG-Na (ID). Compared with the NBW group, the IUGR group showed decreased (P < 0.05) serum and LM antioxidant defense capacity, and increased (P < 0.05) serum and LM damage. Compared with the N group, the I group showed decreased (P < 0.05) MuSCs antioxidant defense capacity, mitochondrial ETC complexes, energy metabolites, and antioxidant defense-related and mitochondrial function-related gene and protein expression levels. The antioxidant defense capacity, mitochondrial ETC complexes, energy metabolites, and antioxidant defense-related and mitochondrial function-related gene and protein expression levels of MuSCs were improved (P < 0.05) in the ID group compared to those in the I group. The MuSCs of IUGR newborns activate the Nrf2/SIRT1/PGC1α network by taking in DMG-Na, thereby neutralizing excessive generated O2•− that may help to improve their unfavorable mitochondrial dysfunction in skeletal muscle.

Assessing Complex I (CI) Mitochondrial Subunit Protein Functions in Candida albicans.

Mitochondria of Candida species play critical roles in cell metabolism and pathogenesis. Greater emphasis in specific mitochondria activities of this fungus have been revealed through studies that defined fungal or Candida-specific subunit proteins of ETC Complexes I, III, and IV (CI, CIII, and CIV). Functional activities of these subunits have been characterized through the construction of single-gene null mutants. Activities common to mitochondria of most eukaryotes include their importance in metabolism, ATP synthesis, oxidative phosphorylation, oxygen consumption, and redox potential. An important difference among specific subunits compared to eukaryotic species is the role of CI fungal-specific subunit proteins in activities specific to Candida albicans, such as cell wall synthesis, especially cell wall mannan and β-glucan synthesis. We have associated cell wall synthesis with a signal transduction pathway that includes a Chk1p fungal-specific pathway. Recently, based upon the specificity of CI subunit specificities, a suggestion is the development of novel antifungals that target mitochondrial activity.

Mechanisms Underlying the Altered Bioenergetics of Aged Brain Microvasculature

Objective Aging impairs the cerebral microvascular function leading to increased risk of stroke and cognitive decline. The objective of this study was to identify the mechanisms underlying the aging-induced alterations in cellular energetics of brain microvessels (BMVs). Methods BMVs were isolated from young (2-4 months) and aged (24 months) male mice (C57Bl/6) using a combination of filters with pore sizes of 300μm and 40μm followed by gradient centrifugation. Extracellular acidification rate (ECAR) and oxygen consumption rate (OCR) were measured from real time ATP rate assay utilizing Seahorse XFe24 analyzer. Subsequently, total ATP production rate, GlycoATP and MitoATP levels were calculated. Mitochondrial fuel dependency of BMVs was assayed. Immunoblotting measured the protein levels of glucose transporter 1 (GLUT1), carnitine palmitoyltransferases (CPTs), 6-phosphofructo-2-kinase/fructose-2, 6-biphosphatase 3 (PKKFB3), and ETC complexes (ETCs). Enzyme assays measured the activity of lactate dehydrogenase (LDH), citrate synthase (CS), and glutamate dehydrogenase (GDH). Results We found that oxidative phosphorylation (OxPhos) is the major contributor of cellular energy in the BMVs instead of glycolysis (67.0±2.1% vs 32.9±2.1%, n=7-10 mice/age group). Aged BMVs exhibited decreased total ATP production (49.7%, 8.8±2.0 vs 17.6±6.7 pico moles of ATP/µg/min, p=0.004, n=7-10 mice/age group). ATP generation from the glycolysis was more diminished in aged BMVs (74.7%, 1.5±0.3 vs 5.9±1.1 pico moles of ATP/µg/min, p=0.004, n=7-10 mice/group) than OxPhos (55.6%, 5.2±0.53 vs 11.7±1.8 pico moles of ATP/µg/min, p=0.01, n=7-10mice/group) when compared with young BMVs. GLUT1 levels were decreased in the aged BMVs when compared with the young BMVs (32%, 0.19±0.01 vs 0.28±0.02 A.U., p=0.01, n=12 mice/group). Glycolysis-stimulating enzyme, PFKFB3 expression was diminished (51.4%, 0.69±0.08 vs 1.42±0.13 A.U., n=6 mice/group) in the aged BMVs. Neither CPT nor ETC protein levels were affected by aging. Activity of Krebs cycle enzyme, CS, was reduced in the aged versus young BMVs (25.7%, 74.3±6.1% vs 100.0±9.9% nanomoles of substrate/min/mg, p=0.04, n=11-12 mice/group). Although relative utilizations of glucose and fatty acids were not affected by aging, glutamine utilization was enhanced in aged BMVs compared to young BMVs which was accompanied by increased activity of GDH, an enzyme involved in the mobilization of glutamine through Krebs cycle (24.7%, 116.8±4.3% vs 93.6±7.3% pico moles of NADH/min/mg, p=0.01, n=11-12 mice/group). Conclusions The results suggest that 1). diminished energy production in the aged BMVs is related to impaired glycolysis and OxPhos, and 2). multiple defects involving the expression of proteins and activity of enzymes related to glycolysis, OxPhos, and energy substrate utilization mediate the aging-induced impaired microvascular bioenergetics.

TNF-\u03b1 differentially modulates subunit levels of respiratory electron transport complexes of ER/PR +ve/\u2212ve breast cancer cells to regulate mitochondrial complex activity and tumorigenic potential

BackgroundTumor necrosis factor-α (TNF-α) is an immunostimulatory cytokine that is consistently high in the breast tumor microenvironment (TME); however, its differential role in mitochondrial functions and cell survival in ER/PR +ve and ER/PR −ve breast cancer cells is not well understood.MethodsIn the current study, we investigated TNF-α modulated mitochondrial proteome using high-resolution mass spectrometry and identified the differentially expressed proteins in two different breast cancer cell lines, ER/PR positive cell line; luminal, MCF-7 and ER/PR negative cell line; basal-like, MDA-MB-231 and explored its implication in regulating the tumorigenic potential of breast cancer cells. We also compared the activity of mitochondrial complexes, ATP, and ROS levels between MCF-7 and MDA-MB-231 in the presence of TNF-α. We used Tumor Immune Estimation Resource (TIMER) webserver to analyze the correlation between TNF-α and mitochondrial proteins in basal and luminal breast cancer patients. Kaplan-Meier method was used to analyze the correlation between mitochondrial protein expression and survival of breast cancer patients.ResultsThe proteome analysis revealed that TNF-α differentially altered the level of critical proteins of mitochondrial respiratory chain complexes both in MCF-7 and MDA-MB-231, which correlated with differential assembly and activity of mitochondrial ETC complexes. The inhibition of the glycolytic pathway in the presence of TNF-α showed that glycolysis is indispensable for the proliferation and clonogenic ability of MDA-MB-231 cells (ER/PR −ve) as compared to MCF-7 cells (ER/PR +ve). The TIMER database showed a negative correlation between the expressions of TNF-α and key regulators of mitochondrial OXPHOS complexes in basal breast vs lobular carcinoma. Conversely, patient survival analysis showed an improved relapse-free survival with increased expression of identified proteins of ETC complexes and survival of the breast cancer patients.ConclusionThe evidence presented in our study convincingly demonstrates that TNF-α regulates the survival and proliferation of aggressive tumor cells by modulating the levels of critical assembly factors and subunits involved in mitochondrial respiratory chain supercomplexes organization and function. This favors the rewiring of mitochondrial metabolism towards anaplerosis to support the survival and proliferation of breast cancer cells. Collectively, the results strongly suggest that TNF-α differentially regulates metabolic adaptation in ER/PR +ve (MCF-7) and ER/PR −ve (MDA-MB-231) cells by modulating the mitochondrial supercomplex assembly and activity.

The Effect of Deflazacort Treatment on the Functioning of Skeletal Muscle Mitochondria in Duchenne Muscular Dystrophy.

Duchenne muscular dystrophy (DMD) is a severe hereditary disease caused by a lack of dystrophin, a protein essential for myocyte integrity. Mitochondrial dysfunction is reportedly responsible for DMD. This study examines the effect of glucocorticoid deflazacort on the functioning of the skeletal-muscle mitochondria of dystrophin-deficient mdx mice and WT animals. Deflazacort administration was found to improve mitochondrial respiration of mdx mice due to an increase in the level of ETC complexes (complexes III and IV and ATP synthase), which may contribute to the normalization of ATP levels in the skeletal muscle of mdx animals. Deflazacort treatment improved the rate of Ca2+ uniport in the skeletal muscle mitochondria of mdx mice, presumably by affecting the subunit composition of the calcium uniporter of organelles. At the same time, deflazacort was found to reduce the resistance of skeletal mitochondria to MPT pore opening, which may be associated with a change in the level of ANT2 and CypD. In this case, deflazacort also affected the mitochondria of WT mice. The paper discusses the mechanisms underlying the effect of deflazacort on the functioning of mitochondria and contributing to the improvement of the muscular function of mdx mice.

Diet with Low Molecular Weight Chitosan exerts neuromodulation in Rotenone induced Drosophila model of Parkinson's disease

Rotenone (ROT) is well known pesticide been used in agriculture due to its low cost and rapid action. This has also been widely used to establish Parkinson's disease (PD) models in rodents. Here we have evaluated the role of well established potent molecule ‘Low Molecular Weight Chitosan (LMWC)’ against ROT induced neurotoxicity in Drosophila melanogaster. Male adult flies (8–10 days) were induced for neurotoxicity using ROT (500 μM) and treated with LMWC (5 & 10 mg/mL basal media) during 7 days experiment. Subsequently, neurodegenerative and behavioural parameters were assessed. As a result, flies exposed to ROT alone showed decreased locomotor behaviour in negative geotaxis and open-field studies and had a higher incidence of mortality compared to control group. These effects were accompanied by decrease in dopamine level, ETC complexes and increase in reactive oxygen species (ROS) production and cholinergic activities in head and body of the flies, suggesting ROT was able to induce oxidative stress. Co-exposure of LMWC reversed the ROT induced locomotor impairment, exploratory deficits and changes the biochemical parameters to normal level and increased the survival rate to 16 days. Thus, we suggested LMWC may be considered as better supplement in the prevention and management of Parkinson's disease.

Aged Diabetic Mice Exhibit Diastolic Dysfuntion Associated with Alterations in Myocardial Mitochondrial Oxidative Phosphorylation Protein Expression and Complex Assemblies

Introduction: Diabetes is associated with greatly increased risk of heart failure and cardiac diastolic dysfunction. In addition, T2D patients exhibit numerous mitochondrial derangements including increased ROS, reduced number, and impaired respiration. Mitochondrial supercomplexes (SC) or respirasomes consist of ETC complexes assembled into discrete functional assemblies to facilitate improved respiration. We hypothesized that T2D myocardium would exhibit reduced SC assembly and altered mitochondrial fission/fusion proteins. Methods: Db/db and wt mice were aged to 12-14 months. Cardiac function was assessed by echocardiography. Mitochondrial SCs as well as indicators of mitochondrial dynamics (OPA1, pDRP1) were determined by BN and SDS-PAGE in isolated mitochondrial. ETC proteins levels were determined by SDS-PAGE in whole cell and mitochondrial fractions. Results: Compared to wt mice, db/db mice had significantly increased indices of diastolic dysfunction including elevated LV mass, increased diastolic volume and dimensions, and E/E’ ratio. T2D mice exhibited a strong trend for reduction in assembly of complex I and IV (64.0+/-5.7 and 59.4+/-3.7 % of wt respectively) into mitochondrial SCs as assessed by BN-PAGE, however this did not reach statistical significance (CI:P=.14 and CIV:P=.09). There were no changes in the levels of complex III. There were no differences in ETC protein expression as determined by SDS-PAGE in equally loaded mitochondrial protein fractions, however there were significant reductions in specific mitochondrial ETC subunits (Ndufa9, SDHA) in whole cell fractions indicating reduced mitochondrial content in db/db mice. Db/db mice had significant reductions in mitochondrial complexes of the fission associated pdrp1 (p=.05), but no changes in either expression or complex formation of the fusion protein Opa1. Conclusion: Diastolic dysfunction in T2D mice is associated with reduced SC assembly and increased markers of fission and reduced mitochondrial content.

Apoptosis inducing factor: Cellular protective function in Dictyostelium discoideum

Apoptosis Inducing Factor (AIF), a nuclear encoded mitochondrial inter-membrane space flavoprotein with intrinsic NADH oxidase activity, plays an important role in inducing cell death mechanisms. In response to cell death signals, it undergoes mitochondrio-nuclear translocation leading to DNA fragmentation. In addition to its role in cell death, AIF has a pro-survival role, wherein it contributes to the maintenance of mitochondrial structure and function in a coordinated manner. However, its exact mechanism of controlling mitochondrial homeostasis is unclear. The current study aims to explore the protective functions of AIF by its downregulation and overexpression in Dictyostelium discoideum. Constitutive AIF downregulated (dR) cells exhibited compromised oxidative phosphorylation along with elevated levels of cellular ROS. Interestingly, constitutive AIF dR cells showed amelioration in the activity of the ETC complexes upon antioxidant treatment, strengthening AIF's role as an ROS regulator, by virtue of its oxidoreductase property. Also, constitutive AIF dR cells showed lower transcript levels of the various subunits of ETC. Moreover, loss of AIF affected mtDNA content and mitochondrial fusion-fission mechanism, which subsequently caused morphometric mitochondrial alterations. Constitutive AIF overexpressed (OE) cells also showed higher cellular ROS and mitochondrial fission genes transcript levels along with reduced mitochondrial fusion genes transcript levels and mtDNA content. Thus, the results of the current study provide a paradigm where AIF is implicated in cell survival by maintaining mitochondrial bioenergetics, morphology and fusion-fission mechanism in D. discoideum, an evolutionarily significant model organism for mitochondrial diseases.

The Role of Adenine Nucleotide Translocase in the Assembly of Respiratory Supercomplexes in Cardiac Cells.

Individual electron transport chain complexes have been shown to assemble into the supramolecular structures known as the respiratory chain supercomplexes (RCS). Several studies reported an associative link between RCS disintegration and human diseases, although the physiological role, structural integrity, and mechanisms of RCS formation remain unknown. Our previous studies suggested that the adenine nucleotide translocase (ANT), the most abundant protein of the inner mitochondrial membrane, can be involved in RCS assembly. In this study, we sought to elucidate whether ANT knockdown (KD) affects RCS formation in H9c2 cardiomyoblasts. Results showed that genetic silencing of ANT1, the main ANT isoform in cardiac cells, stimulated proliferation of H9c2 cardiomyoblasts with no effect on cell viability. ANT1 KD reduced the ΔΨm but increased total cellular ATP levels and stimulated the production of total, but not mitochondrial, reactive oxygen species. Importantly, downregulation of ANT1 had no significant effects on the enzymatic activity of individual ETC complexes I–IV; however, RCS disintegration was stimulated in ANT1 KD cells as evidenced by reduced levels of respirasome, the main RCS. The effects of ANT1 KD to induce RCS disassembly was not associated with acetylation of the exchanger. In conclusion, our study demonstrates that ANT is involved in RCS assembly.

Molecular insights into the role of mitochondria in non-alcoholic fatty liver disease.

Nonalcoholic fatty liver disease (NAFLD) is rapidly becoming the most common cause of fatal liver diseases such as cirrhosis, liver cancer, and indications for orthotopic liver transplantation. Given its high prevalence, the absence of FDA-approved drugs for NAFLD is noticeable. In the pathogenesis of NAFLD, it is well known that mitochondrial dysfunction arises as a result of changes in ETC complexes and the membrane potential (Δψm), as well as decreased ATP synthesis. Due to their fundamental role in energy metabolism and cell death decision, alterations in mitochondria are considered to be critical factors causing NAFLD. Reduced levels of β-oxidation, along with increased lipogenesis, result in lipid accumulation in hepatocytes, and the subsequent production of reactive oxygen species and hepatocyte injury, which contribute to hepatic inflammation and fibrosis through the activations of Kupffer cells and hepatic stellate cells. Here, we review the latest findings describing the involvement of mitochondrial processes in the development of NAFLD and discuss the potential targets against which therapeutics for this disease can be developed.

Low Molecular Weight Chitosan (∼20 kDa) protects acrylamide induced oxidative stress in D. melanogaster by restoring dopamine and KIF5B levels

Acrylamide (AA) presence and formation are predominant in fried, baked and heat-processed foods. Using Drosophila model, we have investigated the dietary AA-arbitrate oxidative stress induced neurotoxicity and the effect of soluble Low Molecular Weight Chitosan (LMWC) supplementation. We assessed the neurodegenerative and behavioural effect of AA (0–10 mM) exposure in Drosophila (adult males). As a result, the exposed flies showed distinctive locomotor impairments and incident of mortality [51% in 5 mM AA (sub-toxic level) for 7 days] and higher mortality with increased concentration of acrylamide. Further, exposure of AA toxicity was also correlated with changing levels of oxidative markers, ETC complexes and cholinergic function of flies. Decreased dopamine (25 μg/mg) and kinesin motor protein levels were confirmed by HPLC and Immunoblotting studies, respectively. Interestingly, the co-exposure of LMWC alongside AA ameliorates respective biochemical changes with restoring dopamine (30 μg/mg, control groups 32 μg/mg) and kinesin motor protein (KIF5B) levels. These results indicated that supplementation of biocompatible LMWC may be promising candidate for complete protection against AA induced oxidative stress.

107: Effects of chronic intrauterine hypoxia on mitochondrial respiratory protein function within fetal guinea pig forebrain

Antenatal intrauterine insults are the predominant cause of neonatal hypoxic ischemic encephalopathy. Neonatal brain MRI studies demonstrate abnormal brain patterns attributed to both preterm and term intrauterine insults coexisting in the same child, showing secondary brain injury developing in fetuses compromised by an earlier intrauterine insult. Altered mitochondrial oxidative phosphorylation and ATP production plays a pivotal role in secondary brain injury. We hypothesize that the degree of mitochondrial dysfunction is dependent on timing of the initial brain injury. Using an animal model of chronic intrauterine hypoxia, we characterized mitochondrial function in the fetal forebrain after early- or late-onset hypoxia by examining the expression and activity of electron transport chain proteins (ETC). Pregnant guinea pigs were exposed to either normoxia (n=12) or hypoxia (ambient O2 10.5%) starting at 28 d (early- onset, n=6) or 50 d (late-onset, n=6) gestation until term (65d). Male fetuses were extracted from anesthetized sows, weighed and forebrains were excised. Protein expression of ETC Complexes I-V (CI-CV) and enzyme activity of Complex I (CI activity) were measured by Western Blot Analysis and Enzyme-Linked Immunoassay, respectively. Results of the hypoxic effects are means +/- SEM, with statistical comparison to normoxia by one-tailed T-Test. Hypoxia reduced fetal body weight and crown rump length while increasing brain:body ratio (Table 1). Late-onset hypoxia did not effect either CI-V expression or CI activity. In contrast, early-onset hypoxia decreased expression of CI (49%) and CII (40%). CIII-V changes were not significant (Fig.1A). CI activity in early hypoxia increased 48% compared to normoxia controls (Fig.1B). The impact of hypoxia on mitochondrial respiratory chain function in the fetal forebrain varied with the gestational age of exposure. Early exposure to significant hypoxia produced an enduring deficit in mitochondrial function exemplified by disruption of CI/II expression and dysregulation of CI activity. This likely mediates ongoing electron leakage, the base cause of oxidative stress, and ongoing brain injury.View Large Image Figure ViewerDownload Hi-res image Download (PPT)

Dual Perturbation of Electron Transport Chain (ETC) Complex and ATP Synthase Triggers PINK1/Parkin‐dependent Mitophagy

Neurodegenerative disorders like Parkinson's Disease arise in part due to accumulation of unhealthy mitochondria. PINK1 and Parkin, found to be deficient in some Familial Parkinson's Disease patients, are known to orchestrate mitochondrial quality control responses like mitophagy. Research into PINK1 and Parkin function has been focused primarily on downstream signaling events, but the exact type of damage signals originating from defected mitochondria to trigger PINK1 stabilization and subsequent Parkin recruitment to the outer membrane of damaged mitochondria remains elusive. We propose PINK1 and Parkin act in tandem to monitor the integrity and activity of ETC complexes and ATP synthase to ensure robust energy production. To test this hypothesis, we profiled several small molecule inhibitors with well‐documented inhibitory activities towards ETC complexes for their effects on PINK1 and Parkin activation. We found that while dual inhibition of complex III and V results in robust Parkin recruitment to damaged mitochondria and mitophagy responses, dual inhibition of complex I and V has only modest effect and other combinations have no effect. PINK1 and Parkin activation as a result of dual assault of ETC complexes can be phenocopied with perturbations of these complexes with siRNA. Thus, our study supports the idea that PINK1 and Parkin are guardians of ETC quality and status. Damaged mitochondria as a result of specific defects in oxidative phosphorylation can be cleared by PINK1 and Parkin dependent mitophagy. This mechanism could be relevant in understanding their dysregulation in Parkinson's Disease.Support or Funding InformationNational Institute of General Medical Sciences R01 GM113141, National Institute of General Medical Sciences T32GM08759, S10 RR026680This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Abstract 21186: Amelioration of Myocardial Stunning in Pigs Undergoing Cp/cpb With Bkca Channel Activator Supplemented Cardioplegia

Introduction: Activation of mitochondrial large conductance Ca++-activated K+ channels (BKCa) is cardioprotective in cell and rodent isolated heart models of cardioplegic arrest and reperfusion. We previously demonstrated cardioprotection is associated with reduced mitochondrial ROS generation and reorganization of individual electron transport chain complexes into physically associated respiratory supercomplexes (SC) made of ETC complex I/III/IV. In this study, we examined the efficacy of BKCa activation during cardioplegic arrest in a clinically relevant large animal study using pigs subjected to CP/CPB and reperfusion. Methods: 50 kg Yorkshire pigs of either sex were subjected to CP/CPB (hyperkalemic cold blood CP, 1 hr) with or without the BKCa activator mallotoxin (MTX (aka rottlerin)) supplied in the CP solution, followed by reperfusion (1hr). Pigs were instrumented with a PV catheter and IVC occlusions performed at baseline and 30 and 60 mins after weening from bypass. Following surgery, mitochondria were isolated and subjected to Blue-Native PAGE and immunoblotting of ETC complexes to visualize large (~100-1500 kD) mitochondrial SC. Results: Baseline hemodynamics were similar between groups. CP/CPB resulted in significant myocardial stunning in pigs as assessed by LVDP (pre 64.8 mmHg vs post 42.0, P&lt;.05 ), +/-dP/dt (pre 971.4 and -911.8 vs post:588.0 and -541.0, P&lt;.05 ), and end-systolic elastance (Ees: pre 1.28 vs post .63, P=0.1 ) at 60 min reperfusion. However, pigs arrested with CP containing MTX (500 nM) exhibited greatly reduced myocardial stunning with significant improvements in LVDP (pre:65.7 post:67.8 mmHg, P&lt;.01 vs post CP/CPB group) +/- dP/dt (pre:1026.0 and -911.8 vs post:1441.7 and -984.8 mmHg/s, P=.02 and .04 vs CP/CPB) and Ees (pre 1.1, post 1.35, P=.02 vs CP/CPB). LV mitochondria isolated from both groups had two major I/III/IV mitochondrial SC’s of ~1300 (SC1) and ~1000 kD (SC2) as assessed by BN-PAGE and immunoblot. The CP/CPB + MTX group had significant increases in both SC1 (6.0 % vs 18.0%, P=.01) and SC2 (19.7% vs 52.3%, P&lt;.01) expressed as percent of total assembled complex IV. Conclusion: Addition of a BKCa activator to CP solutions ameliorates myocardial stunning in large animals subjected to CP/CPB and reperfusion.

Inhibition of poly(ADP-ribose) polymerase-1 alters expression of mitochondria-related genes in PC12 cells: relevance to mitochondrial homeostasis in neurodegenerative disorders

Alzheimer's disease (AD) is characterized by the release of amyloid beta peptides (Aβ) in the form of monomers/oligomers which may lead to oxidative stress, mitochondria dysfunction, synaptic loss, neuroinflammation and, in consequence, to overactivation of poly(ADP-ribose) polymerase-1 (PARP-1). However, Aβ peptides are also released in the brain ischemia, traumatic injury and in inflammatory response. PARP-1 is suggested to be a promising target in therapy of neurodegenerative disorders.We investigated the impact of PARP-1 inhibition on transcription of mitochondria-related genes in PC12 cells. Moreover, the effect of PARP-1 inhibitor (PJ34) on cells subjected to Aβ oligomers (AβO) – evoked stress was analyzed.Our data demonstrated that inhibition of PARP-1 in PC12 cells enhanced the transcription of genes for antioxidative enzymes (Sod1, Gpx1, Gpx4), activated genes regulating mitochondrial fission/fusion (Mfn1, Mfn2, Dnm1l, Opa1, Fis1), subunits of ETC complexes (mt-Nd1, Sdha, mt-Cytb) and modulated expression of several TFs, enhanced Foxo1 and decreased Nrf1, Stat6, Nfkb1. AβO elevated free radicals concentration, decreased mitochondria membrane potential (MMP) and cell viability after 24h. Gene transcription was not affected by AβO after 24h, but was significantly downregulated after 96h. In AβO stress, PJ34 exerted stimulatory effect on expression of several genes (Gpx1, Gpx4, Opa1, Mfn2, Fis1 and Sdha), decreased transcription of numerous TFs (Nrf1, Tfam, Stat3, Stat6, Trp53, Nfkb1) and prevented oxidative stress. Our results indicated that PARP-1 inhibition significantly enhanced transcription of genes involved in antioxidative defense and in regulation of mitochondria function, but was not able to ameliorate cells viability affected by Aβ.