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Driescher 2019 Heliyon + (Although there is evidence linking sugar-s … Although there is evidence linking sugar-sweetened beverage (SSB) intake with the development of cardio-metabolic diseases, the underlying mechanisms remain unclear. The current study therefore evaluated the effects of SSB consumption by establishing a unique in-house ''in vivo'' experimental model.Male Wistar rats were divided into two groups: a) one consuming a popular local SSB (SSB- Jive), and b) a control group (Control-water) for a period of three and six months (n = 6 per group), respectively. Rats were gavaged on a daily basis with an experimental dosage amounting to half a glass per day (in human terms) (SSB vs. water). Cardiac function was assessed at baseline (echocardiography) and following ''ex vivo'' ischemia-reperfusion of the isolated perfused working rat heart. Oral glucose tolerance tests and mitochondrial respiratory analyses were also performed. In addition, the role of non-oxidative glucose pathways (NOGPs), i.e. the polyol pathway, hexosamine biosynthetic pathway (HBP) and PKC were assessed.These data show that SSB intake: a) resulted in increased weight gain, but did not elicit major effects in terms of insulin resistance and cardiac function after three and six months, respectively; b) triggered myocardial NOGP activation after three months with a reversion after six months; and c) resulted in some impairment in mitochondrial respiratory capacity in response to fatty acid substrate supply after six months.SSB intake did not result in cardiac dysfunction or insulin resistance. However, early changes at the molecular level may increase risk in the longer term.evel may increase risk in the longer term.)
Acuña-Castroviejo 2011 Curr Top Med Chem + (Although two main hypotheses of mitochondr … Although two main hypotheses of mitochondrial origin have been proposed, i.e., the autogenous and the endosymbiotic, only the second is being seriously considered currently. The 'hydrogen hypothesis' invokes metabolic symbiosis as the driving force for a symbiotic association between an anaerobic, strictly hydrogen-dependent (the host) and an eubacterium (the symbiont) that was able to respire, but which generated molecular hydrogen as an end product of anaerobic metabolism. The resulting proto-eukaryotic cell would have acquired the essentials of eukaryotic energy metabolism, evolving not only aerobic respiration, but also the physiological cost of the oxygen consumption, i.e., generation of reactive oxygen species (ROS) and the associated oxidative damage. This is not the only price to pay for respiring oxygen: mitochondria possess nitric oxide (NO·) for regulatory purposes but, in some instances it may react with superoxide anion radical to produce the toxic reactive nitrogen species (RNS), i.e. peroxynitrite anion, and the subsequent nitrosative damage. New mitochondria contain their own genome with a modified genetic code that is highly conserved among mammals. The transcription of certain mitochondrial genes may depend on the redox potential of the mitochondrial membrane. Mitochondria are related to the life and death of cells. They are involved in energy production and conservation, having an uncoupling mechanism to produce heat instead of ATP, but they are also involved in programmed cell death. Increasing evidence suggest the participation of mitochondria in neurodegenerative and neuromuscular diseases involving alterations in both nuclear (nDNA) and mitochondrial (mtDNA) DNA. Melatonin is a known powerful antioxidant and anti-inflammatory and increasing experimental and clinical evidence shows its beneficial effects against oxidative/nitrosative stress status, including that involving mitochondrial dysfunction. This review summarizes the data and mechanisms of action of melatonin in relation to mitochondrial pathologies. in relation to mitochondrial pathologies.)
Vicentini 2019 Plant Physiol Biochem + (Aluminum (Al) toxicity has been recognized … Aluminum (Al) toxicity has been recognized to be a main limiting factor of crop productivity in acid soil. Al interacts with cell walls disrupting the functions of the plasma membrane and is associated with oxidative damage and mitochondrial dysfunction. ''Jatropha curcas'' L. (''J. curcas'') is a drought resistant plant, widely distributed around the world, with great economic and medicinal importance. Here we investigated the effects of Al on ''J. curcas'' mitochondrial function and cell viability, analyzing mitochondrial respiration, phenolic compounds, reducing sugars and cell viability in cultured ''J. curcas'' cells. The results showed that at 70 μM, Al limited mitochondrial respiration by inhibiting the alternative oxidase (AOX) pathway in the respiratory chain. An increased concentration of reducing sugars and reduced concentration of intracellular phenolic compounds was observed during respiratory inhibition. After inhibition, a time-dependent upregulation of AOX mRNA was observed followed by restoration of respiratory activity and reducing sugar concentrations. Cultured ''J. curcas'' cells were very resistant to Al-induced cell death. In addition, at 70 μM, Al also appeared as an inhibitor of cell wall invertase. In conclusion, Al tolerance in cultured ''J. curcas'' cells involves a inhibition of mitochondrial AOX pathway, which seems to start an oxidative burst to induce AOX upregulation, which in turn restores consumption of O2 and substrates. These data provide new insight into the signaling cascades that modulate the Al tolerance mechanism.Copyright © 2019 Elsevier Masson SAS. All rights reserved.ght © 2019 Elsevier Masson SAS. All rights reserved.)
Vicentini 2019 Plant Physiol Biochim + (Aluminum (Al) toxicity has been recognized … Aluminum (Al) toxicity has been recognized to be a main limiting factor of crop productivity in acid soil. Al interacts with cell walls disrupting the functions of the plasma membrane and is associated with oxidative damage and mitochondrial dysfunction. ''Jatropha curcas'' L. (J. curcas) is a drought resistant plant, widely distributed around the world, with great economic and medicinal importance. Here we investigated the effects of Al on ''J. curcas'' mitochondrial function and cell viability, analyzing mitochondrial respiration, phenolic compounds, reducing sugars and cell viability in cultured ''J. curcas'' cells. The results showed that at 70 μM, Al limited mitochondrial respiration by inhibiting the alternative oxidase (AOX) pathway in the respiratory chain. An increased concentration of reducing sugars and reduced concentration of intracellular phenolic compounds was observed during respiratory inhibition. After inhibition, a time-dependent upregulation of AOX mRNA was observed followed by restoration of respiratory activity and reducing sugar concentrations. Cultured ''J. curcas'' cells were very resistant to Al-induced cell death. In addition, at 70 μM, Al also appeared as an inhibitor of cell wall invertase. In conclusion, Al tolerance in cultured ''J. curcas'' cells involves a inhibition of mitochondrial AOX pathway, which seems to start an oxidative burst to induce AOX upregulation, which in turn restores consumption of O2 and substrates. These data provide new insight into the signaling cascades that modulate the Al tolerance mechanism. that modulate the Al tolerance mechanism.)
Stojakovic 2021 Commun Biol + (Alzheimer's Disease (AD) is a devastating … Alzheimer's Disease (AD) is a devastating neurodegenerative disorder without a cure. Here we show that mitochondrial respiratory chain complex I is an important small molecule druggable target in AD. Partial inhibition of complex I triggers the AMP-activated protein kinase-dependent signaling network leading to neuroprotection in symptomatic APP/PS1 female mice, a translational model of AD. Treatment of symptomatic APP/PS1 mice with complex I inhibitor improved energy homeostasis, synaptic activity, long-term potentiation, dendritic spine maturation, cognitive function and proteostasis, and reduced oxidative stress and inflammation in brain and periphery, ultimately blocking the ongoing neurodegeneration. Therapeutic efficacy ''in vivo'' was monitored using translational biomarkers FDG-PET, 31P NMR, and metabolomics. Cross-validation of the mouse and the human transcriptomic data from the NIH Accelerating Medicines Partnership-AD database demonstrated that pathways improved by the treatment in APP/PS1 mice, including the immune system response and neurotransmission, represent mechanisms essential for therapeutic efficacy in AD patients.l for therapeutic efficacy in AD patients.)
Lim 2010 Proteomics + (Alzheimer's disease (AD) and type 2 diabet … Alzheimer's disease (AD) and type 2 diabetes mellitus (T2DM) are leading causes of morbidity and mortality in the elderly. Both diseases are characterized by amyloid deposition in target tissues: aggregation of amylin in T2DM is associated with loss of insulin-secreting beta-cells, while amyloid beta (A beta) aggregation in AD brain is associated with neuronal loss. Here, we used quantitative iTRAQ proteomics as a discovery tool to show that both A beta and human amylin (HA) deregulate identical proteins, a quarter of which are mitochondrial, supporting the notion that mitochondrial dysfunction is a common target in these two amyloidoses. A functional validation revealed that mitochondrial complex IV activity was significantly reduced after treatment with either HA or A beta, as was mitochondrial respiration. In comparison, complex I activity was reduced only after treatment with HA. A beta and HA, but not the non-amyloidogenic rat amylin, induced significant increases in the generation of ROS. Co-incubation of HA and A beta did not produce an augmented effect in ROS production, again suggesting common toxicity mechanisms. In conclusion, our data suggest that A beta and HA both exert toxicity, at least in part, via mitochondrial dysfunction, thus restoring their function may be beneficial for both AD and T2DM.on may be beneficial for both AD and T2DM.)
Lim 2011 PloS One + (Alzheimer's disease (AD) is a conformation … Alzheimer's disease (AD) is a conformational disease that is characterized by amyloid-β (Aβ) deposition in the brain. Aβ exerts its toxicity in part by receptor-mediated interactions that cause down-stream protein misfolding and aggregation, as well as mitochondrial dysfunction. Recent reports indicate that Aβ may also interact directly with intracellular proteins such as the mitochondrial enzyme ABAD (Aβ binding alcohol dehydrogenase) in executing its toxic effects. Mitochondrial dysfunction occurs early in AD, and Aβ's toxicity is in part mediated by inhibition of ABAD as shown previously with an ABAD decoy peptide. Here, we employed AG18051, a novel small ABAD-specific compound inhibitor, to investigate the role of ABAD in Aβ toxicity. Using SH-SY5Y neuroblastoma cells, we found that AG18051 partially blocked the Aβ-ABAD interaction in a pull-down assay while it also prevented the Aβ42-induced down-regulation of ABAD activity, as measured by levels of estradiol, a known hormone and product of ABAD activity. Furthermore, AG18051 is protective against Aβ42 toxicity, as measured by LDH release and MTT absorbance. Specifically, AG18051 reduced Aβ42-induced impairment of mitochondrial respiration and oxidative stress as shown by reduced ROS (reactive oxygen species) levels. Guided by our previous finding of shared aspects of the toxicity of Aβ and human amylin (HA), with the latter forming aggregates in Type 2 diabetes mellitus (T2DM) pancreas, we determined whether AG18051 would also confer protection from HA toxicity. We found that the inhibitor conferred only partial protection from HA toxicity indicating distinct pathomechanisms of the two amyloidogenic agents. Taken together, our results present the inhibition of ABAD by compounds such as AG18051 as a promising therapeutic strategy for the prevention and treatment of AD, and suggest levels of estradiol as a suitable read-out.evels of estradiol as a suitable read-out.)
Onukwufor 2022 Antioxidants (Basel) + (Alzheimer's disease (AD) is a devastating … Alzheimer's disease (AD) is a devastating progressive neurodegenerative disease characterized by neuronal dysfunction, and decreased memory and cognitive function. Iron is critical for neuronal activity, neurotransmitter biosynthesis, and energy homeostasis. Iron accumulation occurs in AD and results in neuronal dysfunction through activation of multifactorial mechanisms. Mitochondria generate energy and iron is a key co-factor required for: (1) ATP production by the electron transport chain, (2) heme protein biosynthesis and (3) iron-sulfur cluster formation. Disruptions in iron homeostasis result in mitochondrial dysfunction and energetic failure. Ferroptosis, a non-apoptotic iron-dependent form of cell death mediated by uncontrolled accumulation of reactive oxygen species and lipid peroxidation, is associated with AD and other neurodegenerative diseases. AD pathogenesis is complex with multiple diverse interacting players including Aβ-plaque formation, phosphorylated tau, and redox stress. Unfortunately, clinical trials in AD based on targeting these canonical hallmarks have been largely unsuccessful. Here, we review evidence linking iron dysregulation to AD and the potential for targeting ferroptosis as a therapeutic intervention for AD.osis as a therapeutic intervention for AD.)
De Godoy 2018 J Biol Chem + (Alzheimer's disease (AD) is a disabling an … Alzheimer's disease (AD) is a disabling and highly prevalent neurodegenerative condition, for which there are no effective therapies. Soluble oligomers of the amyloid-β peptide (AβOs) are thought to be proximal neurotoxins involved in early neuronal oxidative stress and synapse damage, ultimately leading to neurodegeneration and memory impairment in AD. The aim of the current study was to evaluate the neuroprotective potential of mesenchymal stem cells (MSCs) against the deleterious impact of AβOs on hippocampal neurons. To this end, we established transwell cocultures of rat hippocampal neurons and MSCs. We show that MSCs and MSC-derived extracellular vesicles protect neurons against AβO-induced oxidative stress and synapse damage, revealed by loss of pre- and postsynaptic markers. Protection by MSCs entails three complementary mechanisms: 1) internalization and degradation of AβOs; 2) release of extracellular vesicles containing active catalase; and 3) selective secretion of interleukin-6, interleukin-10, and vascular endothelial growth factor to the medium. Results support the notion that MSCs may represent a promising alternative for cell-based therapies in AD.lternative for cell-based therapies in AD.)
Martins 2017 Thesis + (Alzheimer's disease (AD) is a neurological … Alzheimer's disease (AD) is a neurological disorder that affects a large part of the world's population. It is characterized by memory loss, progressive dementia, behavioral changes and inability to perform routine activities. Histopathologically, AD presents the formation of plaques by the aggregation of amyloid-β peptides (Aβ) and neurofibrillary tangles, due to hyperphosphorylation of tau protein. The aim of this study was to investigate alterations in gene and protein expression related to AD and to evaluate the mechanisms involved in initial events of Aβ1-40 peptide toxicity after intracerebroventricular (i.c.v.) infusion. In addition, it was evaluated whether pre-treatment with atorvastatin prevents the toxic effects of this peptide. Adult male Swiss albino mice (3 months / 40-50g) were treated with atorvastatin 10 mg / kg / day, orally, or vehicle (0.9% saline) for 7 days. On the seventh day the aggregate form of Aβ1-40 (i.c.v., 400pmol / site) or saline was administered. After 24h, the animals were euthanized for biochemical analysis. The results show that atorvastatin is able to prevent the gene expression reduction of the postsynaptic protein PSD-95, the NMDA receptor GluN1 subunit and glutamatergic transporters GLAST and GLT-1 induced by Aβ1-40 infusion in the hippocampus. Aβ promoted a decrease in BDNF expression and an increase in reactive oxygen (ROS) and nitrogen (RNS) species levels. Pretreatment with atorvastatin was able to prevent the increased ROS and RNS. Through the evaluation of mitochondrial functionality by high resolution respirometry, we observed that Aβ1-40 did not significantly alter parameters of oxygen consumption. However, atorvastatin increased the mitochondrial respiratory capacity assessed in hippocampal homogenates. In conclusion, we observed that Aβ1-40 toxicity presents as initial events changes in proteins related to glutamatergic neurotransmission and oxidative stress. Atorvastatin prevents initial oxidative stress and increases mitochondrial respiratory capacity by an as yet unknown action mechanism, requiring further studies.s mitochondrial respiratory capacity by an as yet unknown action mechanism, requiring further studies.)
Rhein 2009 Proc Natl Acad Sci U S A + (Alzheimer's disease (AD) is characterized … Alzheimer's disease (AD) is characterized by amyloid-beta (Abeta)-containing plaques, neurofibrillary tangles, and neuron and synapse loss. Tangle formation has been reproduced in P301L tau transgenic pR5 mice, whereas APPswPS2N141I double-transgenic APP152 mice develop Abeta plaques. Cross-breeding generates triple transgenic (tripleAD) mice that combine both pathologies in one model. To determine functional consequences of the combined Abeta and tau pathologies, we performed a proteomic analysis followed by functional validation. Specifically, we obtained vesicular preparations from tripleAD mice, the parental strains, and nontransgenic mice, followed by the quantitative mass-tag labeling proteomic technique iTRAQ and mass spectrometry. Within 1,275 quantified proteins, we found a massive deregulation of 24 proteins, of which one-third were mitochondrial proteins mainly related to complexes I and IV of the oxidative phosphorylation system (OXPHOS). Notably, deregulation of complex I was tau dependent, whereas deregulation of complex IV was Abeta dependent, both at the protein and activity levels. Synergistic effects of Abeta and tau were evident in 8-month-old tripleAD mice as only they showed a reduction of the mitochondrial membrane potential at this early age. At the age of 12 months, the strongest defects on OXPHOS, synthesis of ATP, and reactive oxygen species were exhibited in the tripleAD mice, again emphasizing synergistic, age-associated effects of Abeta and tau in perishing mitochondria. Our study establishes a molecular link between Abeta and tau protein in AD pathology in vivo, illustrating the potential of quantitative proteomics.link between Abeta and tau protein in AD pathology in vivo, illustrating the potential of quantitative proteomics.)
Grimm 2016 Methods Mol Biol + (Alzheimer's disease (AD) is characterized … Alzheimer's disease (AD) is characterized by the presence of amyloid plaques (aggregates of amyloid-β [Aβ]) and neurofibrillary tangles (aggregates of tau) in the brain, but the underlying mechanisms of the disease are still partially unclear. A growing body of evidence supports mitochondrial dysfunction as a prominent and early, chronic oxidative stress-associated event that contributes to synaptic abnormalities, and, ultimately, selective neuronal degeneration in AD. Using a high-resolution respirometry system, we shed new light on the close interrelationship of this organelle with Aβ and tau in the pathogenic process underlying AD by showing a synergistic effect of these two hallmark proteins on the oxidative phosphorylation capacity of mitochondria isolated from the brain of transgenic AD mice. In the present chapter, we first introduce the principle of the Aβ and tau interaction on mitochondrial respiration, and secondly, we describe in detail the used respiratory protocol.e in detail the used respiratory protocol.)
Leuner 2012 Mol Neurobiol + (Alzheimer's disease (AD) is the most commo … Alzheimer's disease (AD) is the most common progressive neurodegenerative disease. Today, AD affects millions of people worldwide and the number of AD cases will increase with increased life expectancy. The AD brain is marked by severe neurodegeneration like the loss of synapses and neurons, atrophy and depletion of neurotransmitter systems in the hippocampus and cerebral cortex. Recent findings suggest that these pathological changes are causally induced by mitochondrial dysfunction and increased oxidative stress. These changes are not only observed in the brain of AD patients but also in the periphery. In this review, we discuss the potential role of elevated apoptosis, increased oxidative stress and especially mitochondrial dysfunction as peripheral markers for the detection of AD in blood cells especially in lymphocytes. We discuss recent not otherwise published findings on the level of complex activities of the respiratory chain comprising mitochondrial respiration and the mitochondrial membrane potential (MMP). We obtained decreased basal MMP levels in lymphocytes from AD patients as well as enhanced sensitivity to different complex inhibitors of the respiratory chain. These changes are in line with mitochondrial defects obtained in AD cell and animal models, and in post-mortem AD tissue. Importantly, these mitochondrial alterations where not only found in AD patients but also in patients with mild cognitive impairment (MCI). These new findings point to a relevance of mitochondrial function as an early peripheral marker for the detection of AD and MCI.al marker for the detection of AD and MCI.)
Djordjevic 2017 Neuroscience + (Alzheimer's disease (AD) is the most commo … Alzheimer's disease (AD) is the most common late onset neurodegenerative disorder with indications that women are disproportionately affected. Mitochondrial dysfunction has been one of the most discussed hypotheses associated with the early onset and progression of AD, and it has been attributed to intraneuronal accumulation of amyloid β (Aβ). It was suggested that one of the possible mediators for Aβ-impaired mitochondrial function is the nuclear factor kappa B (NF-κB) signaling pathway. NF-κB plays important roles in brain inflammation and antioxidant defense, as well as in the regulation of mitochondrial function, and studies have confirmed altered NF-κB signaling in AD brain. In this study, we looked for sex-based differences in impaired bioenergetic processes and NF-κB signaling in the AD-like brain using transgenic (Tg) CRND8 mice that express excessive brain Aβ, but without tau pathology. Our results show that mitochondrial dysfunction is not uniform in affected brain regions. We observed increased basal and coupled respiration in the hippocampus of TgCRND8 females only, along with a decreased Complex II-dependent respiratory activity. Cortical mitochondria from TgCRND8 mice have reduced uncoupled respiration capacity, regardless of sex. The pattern of changes in NF-κB signaling was the same in both brain structures, but was sex specific. Whereas in females there was an increase in all three subunits of NF-κB, in males we observed increase in p65 and p105, but no changes in p50 levels. These results demonstrate that mitochondrial function and inflammatory signaling in the AD-like brain is region- and sex-specific, which is an important consideration for therapeutic strategies. consideration for therapeutic strategies.)
Silaidos 2018 Biol Sex Differ + (Alzheimer's disease (AD) is the most commo … Alzheimer's disease (AD) is the most common form of dementia, and it affects more women than men. Mitochondrial dysfunction (MD) plays a key role in AD, and it is detectable at an early stage of the degenerative process in peripheral tissues, such as peripheral mononuclear blood cells (PBMCs). However, whether these changes are also reflected in cerebral energy metabolism and whether sex-specific differences in mitochondrial function occur are not clear. Therefore, we estimated the correlation between mitochondrial function in PBMCs and brain energy metabolites and examined sex-specific differences in healthy participants to elucidate these issues.The current pilot study included 9 male and 15 female healthy adults (mean age 30.8 ± 7.1 years). Respiration and activity of mitochondrial respiratory complexes were measured using a Clark-electrode (Oxygraph-2k system), and adenosine triphosphate (ATP) levels were determined using a bioluminescence-based assay in isolated PBMCs. Citrate synthase activity as a mitochondrial marker was measured using a photometric assay. Concentrations of brain energy metabolites were quantified in the same individuals using 1H-magnetic resonance spectroscopy (MRS).We detected sex-associated differences in mitochondrial function. Mitochondrial complexes I, I+II, and IV and uncoupled respiration and electron transport system (ETS) capacity in PBMCs isolated from blood samples of females were significantly (p < 0.05; p < 0.01) higher compared to males. ATP levels in the PBMCs of female participants were approximately 10% higher compared to males. Citrate synthase (CS) activity, a marker of mitochondrial content, was significantly (p < 0.05) higher in females compared to males. Sex-associated differences were also found for brain metabolites. The N-acetylaspartate (NAA) concentration was significantly higher in female participants compared to males in targeted regions. This difference was observed in white matter (WM) and an area with a high percentage (> 50%) of gray matter (GM) (p < 0.05; p < 0.01). The effect sizes indicated a strong influence of sex on these parameters. Sex-associated differences were found in PBMCs and brain, but the determined parameters were not significantly correlated.Our study revealed sex-associated differences in mitochondrial function in healthy participants. The underlying mechanisms must be elucidated in more detail, but our study suggests that mitochondrial function in PBMCs is a feasible surrogate marker to detect differences in mitochondrial function and energy metabolism in humans and it underscores the necessity of sex-specific approaches in therapies that target mitochondrial dysfunction.proaches in therapies that target mitochondrial dysfunction.)
Sharma 2021 Int J Mol Sci + (Alzheimer's disease (AD) is the most frequ … Alzheimer's disease (AD) is the most frequent cause of age-related neurodegeneration and cognitive impairment, and there are currently no broadly effective therapies. The underlying pathogenesis is complex, but a growing body of evidence implicates mitochondrial dysfunction as a common pathomechanism involved in many of the hallmark features of the AD brain, such as formation of amyloid-beta (Aβ) aggregates (amyloid plaques), neurofibrillary tangles, cholinergic system dysfunction, impaired synaptic transmission and plasticity, oxidative stress, and neuroinflammation, that lead to neurodegeneration and cognitive dysfunction. Indeed, mitochondrial dysfunction concomitant with progressive accumulation of mitochondrial Aβ is an early event in AD pathogenesis. Healthy mitochondria are critical for providing sufficient energy to maintain endogenous neuroprotective and reparative mechanisms, while disturbances in mitochondrial function, motility, fission, and fusion lead to neuronal malfunction and degeneration associated with excess free radical production and reduced intracellular calcium buffering. In addition, mitochondrial dysfunction can contribute to amyloid-β precursor protein (APP) expression and misprocessing to produce pathogenic fragments (e.g., Aβ1-40). Given this background, we present an overview of the importance of mitochondria for maintenance of neuronal function and how mitochondrial dysfunction acts as a driver of cognitive impairment in AD. Additionally, we provide a brief summary of possible treatments targeting mitochondrial dysfunction as therapeutic approaches for AD.function as therapeutic approaches for AD.)
Litwiniuk 2021 Pharmaceuticals (Basel) + (Alzheimer's disease and Parkinson's diseas … Alzheimer's disease and Parkinson's disease are the most common forms of neurodegenerative illnesses. It has been widely accepted that neuroinflammation is the key pathogenic mechanism in neurodegeneration. Both mitochondrial dysfunction and enhanced NLRP3 (nucleotide-binding oligomerization domain (NOD)-like receptor protein 3) inflammasome complex activity have a crucial role in inducing and sustaining neuroinflammation. In addition, mitochondrial-related inflammatory factors could drive the formation of inflammasome complexes, which are responsible for the activation, maturation, and release of pro-inflammatory cytokines, including interleukin-1β (IL-1β) and interleukin-18 (IL-18). The present review includes a broadened approach to the role of mitochondrial dysfunction resulting in abnormal NLRP3 activation in selected neurodegenerative diseases. Moreover, we also discuss the potential mitochondria-focused treatments that could influence the NLRP3 complex.ts that could influence the NLRP3 complex.)
Mezeiova 2020 Eur J Med Chem + (Alzheimer's disease is a progressive brain … Alzheimer's disease is a progressive brain disorder with characteristic symptoms and several pathological hallmarks. The concept of "one drug, one target" has not generated any new drugs since 2004. The new era of drug development in the field of AD builds upon rationally designed multi-target directed ligands that can better address the complexity of AD. Herewith, we designed ten novel derivatives of 2-propargylamino-naphthoquinone. The biological evaluation of these compounds includes inhibition of monoamine oxidase A/B, inhibition of amyloid-beta aggregation, radical-scavenging, and metal-chelating properties. Some of the compounds possess low cytotoxicity profile with an anti-inflammatory ability in the lipopolysaccharide-stimulated cellular model. All these features warrant their further testing in the field of AD. their further testing in the field of AD.)
Bobba 2015 Apoptosis + (Alzheimer’s disease (AD) and cancer procee … Alzheimer’s disease (AD) and cancer proceedvia one or more common molecular mechanisms: a metabolicshift from oxidative phosphorylation to glycolysis—corresponding to the activation of the Warburg effect—occurs in both diseases. The findings reported in this paperdemonstrate that, in the early phase of apoptosis, glucosemetabolism is enhanced, i.e. key proteins which internalizeand metabolize glucose—glucose transporter, hexokinaseand phosphofructokinase—are up-regulated, in concomitancewith a parallel decrease in oxygen consumption bymitochondria and increase of L-lactate accumulation.Reversal of the glycolytic phenotype occurs in the presenceof dichloroacetate, inhibitor of the pyruvate dehydrogenasekinase enzyme, which speeds up apoptosis of cerebellargranule cells, reawakening mitochondria and then modulatingglycolytic enzymes. Loss of the adaptive advantageafforded by aerobic glycolysis, which occurs in the latephase of apoptosis, exacerbates the pathological processesunderlying neurodegeneration, leading inevitably the cellto death. In conclusion, the data propose that both aerobic,i.e. Warburg effect, essentially due to the protectivenumbness of mitochondria, and anaerobic glycolysis, ratherdue to the mitochondrial impairment, characterize theentire time frame of apoptosis, from the early to the latephase, which mimics the development of AD.phase, which mimics the development of AD.)
Integrative Physiology of Exercise 2020 Virtual Event + (American Physiological Society’s (APS)Integrative Physiology of Exercise (IPE) 2020 conference, Virtual Event, 2020)
Petiz 2018 Chem Biol Interact + (Amidines are chemically characterized by t … Amidines are chemically characterized by the presence of two nitrogen atoms that bind to the same carbon atom in its structure. Several biological activities have been ascribed to these compounds. Pentamidine, an aromatic diamidine, is effective in the treatment against ''Pneumocystis carinii'' and leishmaniasis, but it can also have severe side effects. New amidine derivatives have been synthesized, among them N,N'-diphenyl-4-methoxy-benzamidine (methoxyamidine), which is effective against ''Leishmania amazonensis'' (LD50 = 20 μM) and ''Trypanosoma cruzi'' (LD50 = 59 nM). In the present study, methoxyamidine toxicity was evaluated in isolated rat liver mitochondria at the same range of concentrations that exert antiprotozoal activity. In these organelles, actively oxidizing glutamate + malate inhibited state 3 respiration (25 nmol mg-1 of protein) by ∼15%. The sites of inhibition in the respiratory chain were complex I and the segment between ubiquinone and complex III. Methoxyamidine also stimulated state 4 respiration by ∼32% and ∼43% at 50 and 65 nmol mg-1 of protein, respectively. Its uncoupling effect was confirmed by a dose-dependent increase in oxygen consumption in state 4 respiration that was induced by oligomycin, reaching up to ∼69% (65 nmol mg-1 of protein) and an increase in ATPase activity in intact mitochondria by ∼27% and ∼83% at 50 and 65 nmol mg-1 protein, respectively. Swelling that was supported by the oxidation of glutamate + malate in the presence of sodium acetate was reduced by methoxyamidine by ∼16% and 32% at 50 and 65 nmol mg-1 protein, respectively. Mitochondrial swelling in the absence of substrate and in the presence of K+ and valinomycin was inhibited by ∼20% at the same concentrations, suggesting that methoxyamidine affects mitochondrial membrane permeability and fluidity. Our data show that methoxyamidine has slight effects on the energy-linked functions of isolated mitochondria at concentrations that correspond to the LD50 against ''Leishmania amazonensis'' and ''Trypanosoma cruzi''. These findings may prompt further studies that evaluate methoxyamidine toxicity ''in vivo''.nst ''Leishmania amazonensis'' and ''Trypanosoma cruzi''. These findings may prompt further studies that evaluate methoxyamidine toxicity ''in vivo''.)
Hoefler 2016 Physiol Plant + (Amino acid catabolism is essential for adj … Amino acid catabolism is essential for adjusting pool sizes of free amino acids and takes part in energy production as well as nutrient remobilization. The carbon skeletons are generally converted to precursors or intermediates of the tricarboxylic acid cycle. In the case of cysteine, the reduced sulfur derived from the thiol group also has to be oxidized in order to prevent accumulation to toxic concentrations. Here we present a mitochondrial sulfur catabolic pathway catalyzing the complete oxidation of l-cysteine to pyruvate and thiosulfate. After transamination to 3-mercaptopyruvate, the sulfhydryl group from l-cysteine is transferred to glutathione by sulfurtransferase 1 and oxidized to sulfite by the sulfur dioxygenase ETHE1. Sulfite is then converted to thiosulfate by addition of a second persulfide group by sulfurtransferase 1. This pathway is most relevant during early embryo development and for vegetative growth under light-limiting conditions. Characterization of a double mutant produced from ''Arabidopsis thaliana'' T-DNA insertion lines for ETHE1 and sulfurtransferase 1 revealed that an intermediate of the ETHE1 dependent pathway, most likely a persulfide, interferes with amino acid catabolism and induces early senescence.© 2016 Scandinavian Plant Physiology Society.016 Scandinavian Plant Physiology Society.)
Girard 2018 mSphere + (Amino acids participate in several critica … Amino acids participate in several critical processes in the biology of trypanosomatids, such as osmoregulation, cell differentiation, and host cell invasion. Some of them provide reducing power for mitochondrial ATP synthesis. It was previously shown that alanine, which is formed mainly by the amination of pyruvate, is a metabolic end product formed when parasites are replicating in a medium rich in glucose and amino acids. It was shown as well that this amino acid can also be used for the regulation of cell volume and resistance to osmotic stress. In this work, we demonstrate that, despite it being an end product of its metabolism, ''Trypanosoma cruzi'' can take up and metabolize l-Ala through a low-specificity nonstereoselective active transport system. The uptake was dependent on the temperature in the range between 10 and 40°C, which allowed us to calculate an activation energy of 66.4 kJ/mol and estimate the number of transporters per cell at ~436,000. We show as well that, once taken up by the cells, l-Ala can be completely oxidized to CO2, supplying electrons to the electron transport chain, maintaining the electrochemical proton gradient across the mitochondrial inner membrane, and supporting ATP synthesis in ''T. cruzi'' epimastigotes. Our data demonstrate a dual role for Ala in the parasite's bioenergetics, by being a secreted end product of glucose catabolism and taken up as nutrient for oxidative mitochondrial metabolism.It is well known that trypanosomatids such as the etiological agent of Chagas' disease, ''Trypanosoma cruzi'', produce alanine as a main end product of their energy metabolism when they grow in a medium containing glucose and amino acids. In this work, we investigated if under starvation conditions (which happen during the parasite life cycle) the secreted alanine could be recovered from the extracellular medium and used as an energy source. Herein we show that indeed, in parasites submitted to metabolic stress, this metabolite can be taken up and used as an energy source for ATP synthesis, allowing the parasite to extend its survival under starvation conditions. The obtained results point to a dual role for Ala in the parasite's bioenergetics, by being a secreted end product of glucose catabolism and taken up as nutrient for oxidative mitochondrial metabolism.up as nutrient for oxidative mitochondrial metabolism.)
Guitart 2010 J Biol Chem + (Aminoacyl-tRNA synthetases (ARS) are modul … Aminoacyl-tRNA synthetases (ARS) are modular enzymes that aminoacylate transfer RNAs (tRNA) for their use by the ribosome during protein synthesis. ARS are essential and universal components of the genetic code that were almost completely established before the appearance of the last common ancestor of all living species. This long evolutionary history explains the growing number of functions being discovered for ARS, and for ARS homologues, beyond their canonical role in gene translation. Here we present a previously uncharacterized paralogue of seryl-tRNA synthetase named SLIMP (seryl-tRNA synthetase-like insect mitochondrial protein). SLIMP is the result of a duplication of a mitochondrial seryl-tRNA synthetase (SRS) gene that took place in early metazoans and was fixed in Insecta. Here we show that SLIMP is localized in the mitochondria, where it carries out an essential function that is unrelated to the aminoacylation of tRNA. The knockdown of SLIMP by RNA interference (RNAi) causes a decrease in respiration capacity and an increase in mitochondrial mass in the form of aberrant mitochondria.mass in the form of aberrant mitochondria.)
Kumar 2021 JCI Insight + (Ammonia is a cytotoxic metabolite with ple … Ammonia is a cytotoxic metabolite with pleiotropic molecular and metabolic effects, including senescence induction. During dysregulated ammonia metabolism, which occurs in chronic diseases, skeletal muscle becomes a major organ for nonhepatocyte ammonia uptake. Muscle ammonia disposal occurs in mitochondria via cataplerosis of critical intermediary metabolite α-ketoglutarate, a senescence-ameliorating molecule. Untargeted and mitochondrially targeted data were analyzed by multiomics approaches. These analyses were validated experimentally to dissect the specific mitochondrial oxidative defects and functional consequences, including senescence. Responses to ammonia lowering in myotubes and in hyperammonemic portacaval anastomosis rat muscle were studied. Whole-cell transcriptomics integrated with whole-cell, mitochondrial, and tissue proteomics showed distinct temporal clusters of responses with enrichment of oxidative dysfunction and senescence-related pathways/proteins during hyperammonemia and after ammonia withdrawal. Functional and metabolic studies showed defects in electron transport chain complexes I, III, and IV; loss of supercomplex assembly; decreased ATP synthesis; increased free radical generation with oxidative modification of proteins/lipids; and senescence-associated molecular phenotype-increased β-galactosidase activity and expression of p16INK, p21, and p53. These perturbations were partially reversed by ammonia lowering. Dysregulated ammonia metabolism caused reversible mitochondrial dysfunction by transcriptional and translational perturbations in multiple pathways with a distinct skeletal muscle senescence-associated molecular phenotype.senescence-associated molecular phenotype.)

(Ammonia is a cytotoxic metabolite with pleiotropic molecular and metabol)

Chowdhury 2000 Clin Chim Acta + (Amniocytes represent a population of foeta … Amniocytes represent a population of foetal cells that can be used for prenatal diagnosis in families with suspected mitochondrial oxidative phosphorylation (OXPHOS) defects. In this paper, we present a complex protocol for evaluation of the function of mitochondrial OXPHOS enzymes in cultured amniocytes using three independent and complementary methods: (a) spectrophotometry as a tool for determination of the capacities of mitochondrial respiratory-chain enzymes (NADH ubiquinone oxidoreductase, succinate- and glycerophosphate cytochrome c reductase, cytochrome c oxidase and citrate synthase); (b) polarography as a tool for the evaluation of mitochondrial OXPHOS enzyme functions in situ using digitonin-permeabilised amniocytes (rotenone-sensitive oxidation of pyruvate+malate, antimycin A-sensitive oxidation of succinate, KCN-sensitive oxidation of cytochrome c, ADP-activated substrate oxidation) and (c) cytofluorometric determination of tetramethyl rhodamine methyl ester (TMRM) fluorescence in digitonin-permeabilised amniocytes as a sensitive way to determine the mitochondrial membrane potential under steady-state conditions (state 4 with succinate). These protocols are presented together with reference control values using 9–22 independent cultures of amniocytes.g 9–22 independent cultures of amniocytes.)
Pozenel 2019 Cells + (Amniotic cells show exciting stem cell fea … Amniotic cells show exciting stem cell features, which has led to the idea of using living cells of human amniotic membranes (hAMs) in toto for clinical applications. However, under common cell culture conditions, viability of amniotic cells decreases rapidly, whereby reasons for this decrease are unknown so far. Recently, it has been suggested that loss of tissue tension ''in vivo'' leads to apoptosis. Therefore, the aim of this study was to investigate the effect of tissue distention on the viability of amniotic cells ''in vitro''. Thereby, particular focus was put on vital mitochondria-linked parameters, such as respiration and ATP synthesis. Biopsies of hAMs were incubated for 7-21 days either non-distended or distended. We observed increased B-cell lymphoma 2-associated X protein (BAX)/B-cell lymphoma (BCL)-2 ratios in non-distended hAMs at day seven, followed by increased caspase 3 expression at day 14, and, consequently, loss of viability at day 21. In contrast, under distention, caspase 3 expression increased only slightly, and mitochondrial function and cellular viability were largely maintained. Our data suggest that a mechano-sensing pathway may control viability of hAM cells by triggering mitochondria-mediated apoptosis upon loss of tension ''in vitro''. Further studies are required to elucidate the underlying molecular mechanisms between tissue distention and viability of hAM cells.sue distention and viability of hAM cells.)
Antona 2023 Cell Death Discov + (Among all cancers, colorectal cancer (CRC) … Among all cancers, colorectal cancer (CRC) is the 3rd most common and the 2nd leading cause of death worldwide. New therapeutic strategies are required to target cancer stem cells (CSCs), a subset of tumor cells highly resistant to present-day therapy and responsible for tumor relapse. CSCs display dynamic genetic and epigenetic alterations that allow quick adaptations to perturbations. Lysine-specific histone demethylase 1A (KDM1A also known as LSD1), a FAD-dependent H3K4me1/2 and H3K9me1/2 demethylase, was found to be upregulated in several tumors and associated with a poor prognosis due to its ability to maintain CSCs staminal features. Here, we explored the potential role of KDM1A targeting in CRC by characterizing the effect of KDM1A silencing in differentiated and CRC stem cells (CRC-SCs). In CRC samples, KDM1A overexpression was associated with a worse prognosis, confirming its role as an independent negative prognostic factor of CRC. Consistently, biological assays such as methylcellulose colony formation, invasion, and migration assays demonstrated a significantly decreased self-renewal potential, as well as migration and invasion potential upon KDM1A silencing. Our untargeted multi-omics approach (transcriptomic and proteomic) revealed the association of KDM1A silencing with CRC-SCs cytoskeletal and metabolism remodeling towards a differentiated phenotype, supporting the role of KDM1A in CRC cells stemness maintenance. Also, KDM1A silencing resulted in up-regulation of miR-506-3p, previously reported to play a tumor-suppressive role in CRC. Lastly, loss of KDM1A markedly reduced 53BP1 DNA repair foci, implying the involvement of KDM1A in the DNA damage response. Overall, our results indicate that KDM1A impacts CRC progression in several non-overlapping ways, and therefore it represents a promising epigenetic target to prevent tumor relapse.pigenetic target to prevent tumor relapse.)
Gnaiger 2013 Abstract Mito2013 + (Among all humans, the Polar Inuit of Thule … Among all humans, the Polar Inuit of Thule and Qaarnaak in Greenland are the northernmost population, limited to 302 in 1950 and dwindling to 180 in 2004. This human heritage of a culture and physiological type is endangered not only by a historical politically forced limitation of their territory, but by the current effects of global environmental pollution and climate change, causing social destabilization and a shift towards an unhealthy sedentary life style in contrast to the traditional active life style of Inuit hunters. 10 years ago the uncoupling hypothesis was presented for mitochondrial haplogroups of arctic populations suggesting that lower coupling of mitochondrial respiration to ATP production was selected for in favour of higher heat dissipation as an adaptation to cold climates [1,2]. It has been hypothesized that climatic pressures exerted selection for mitochondrial haplogroups in arctic populations as an adaptation to the cold, by increasing heat production through a higher mitochondrial proton leak. We studied muscle mitochondrial function in traditional Inuit hunters from Qaarnaak, Northern Greenland and sedentary Caucasian Danes who engaged in a 42 day ski sojourn across the polar ice caps (80-82o latitude). Small muscle biopsies were obtained from the leg (vastus lateralis) and arm (deltoid) muscles in both Inuit and Danes and mitochondrial function was assessed by high-resolution respirometry [3,4]. OXPHOS capacity in the leg was lower in Inuit compared to Danes consistent with differences in mitochondrial density. Nonetheless, Inuit had a higher OXPHOS capacity with fat substrate in both leg and arm muscles. LEAK respiration was proportionate with OXPHOS such that coupling control was equivalent between groups and across muscles of both arm and leg. After 42 days of skiing Danes demonstrated adaptive substrate control through an increase in fatty acid oxidation towards levels of the Inuit. Biochemical coupling efficiency was preserved across variations in mtDNA, muscle fibre type, uncoupling protein-3 content, muscle OXPHOS capacity, leg and arm muscle, and acclimatization level. This study refutes the hypothesis that uncoupling is higher in skeletal muscle of arctic haplotype populations and reveals that mitochondrial coupling control is tightly conserved across haplotype groups and training status despite large adaptive capacities for substrate oxidation.aptive capacities for substrate oxidation.)
Francisco 2018 J Neurochem + (Among mitochondrial NADP-reducing enzymes, … Among mitochondrial NADP-reducing enzymes, nicotinamide nucleotide transhydrogenase (NNT) establishes an elevated matrix NADPH/NADP+ by catalyzing the reduction of NADP+ at the expense of NADH oxidation coupled to inward proton translocation across the inner mitochondrial membrane. Here, we characterize NNT activity and mitochondrial redox balance in the brain using a congenic mouse model carrying the mutated ''Nnt'' gene from the C57BL/6J strain. The absence of NNT activity resulted in lower total NADPH sources activity in the brain mitochondria of young mice, an effect that was partially compensated in aged mice. Nonsynaptic mitochondria showed higher NNT activity than synaptic mitochondria. In the absence of NNT, an increased release of H2O2 from mitochondria was observed when the metabolism of respiratory substrates occurred with restricted flux through relevant mitochondrial NADPH sources or when respiratory complex I was inhibited. In accordance, mitochondria from ''Nnt''-/- brains were unable to sustain NADP in its reduced state when energized in the absence of carbon substrates, an effect aggravated after H2O2 bolus metabolism. These data indicate that the lack of NNT in brain mitochondria impairs peroxide detoxification, but peroxide detoxification can be partially counterbalanced by concurrent NADPH sources depending on substrate availability. Notably, only brain mitochondria from ''Nnt''-/- mice chronically fed a high-fat diet exhibited lower activity of the redox-sensitive aconitase, suggesting that brain mitochondrial redox balance requires NNT under the metabolic stress of a high-fat diet. Overall, the role of NNT in the brain mitochondria redox balance especially comes into play under mitochondrial respiratory defects or high-fat diet. This article is protected by copyright. All rights reserved.ay under mitochondrial respiratory defects or high-fat diet. This article is protected by copyright. All rights reserved.)
Sharma 2023 Biosci Biotechnol Biochem + (Among the branched chain amino acids (BCAA … Among the branched chain amino acids (BCAAs), leucine and isoleucine have been well-studied for their roles in improving mitochondrial function and reducing oxidative stress. However, role of valine in mitochondrial function regulation and oxidative stress management remains elusive. This study investigated valine effect on mitochondrial function and oxidative stress ''in vitro''. Valine increased expression of genes involved in mitochondrial biogenesis and dynamics. It upregulates mitochondrial function at Complexes I, II and IV levels of electron transport chain. Flow cytometry studies revealed, valine reduced oxidative stress by significantly lowering mitochondrial reactive oxygen species (ROS) and protein expression of 4 hydroxynonenal. Functional role of valine against oxidative stress was analyzed by XFe96 Analyzer. Valine sustained oxidative phosphorylation and improved ATP generation rates during oxidative stress. In conclusion, our findings shed more light on the critical function of valine in protecting mitochondrial function thereby preventing mitochondrial/cellular damage induced by oxidative stress.llular damage induced by oxidative stress.)
Foriel 2015 Abstract MiP2015 + (Among the wide range of mitochondrial diso … Among the wide range of mitochondrial disorders, defects in the oxidative phosphorylation (OxPhos) are the most prevalent. OxPhos deficiencies often lead to early death and are associated with severe and highly variable clinical symptoms. Despite intense efforts in the comprehension of the mechanisms underlying mitochondrial disorders, patients are still without effective treatment. The need of predictive ''in vivo'' models of the pathology is an important issue in the development of new therapeutics in order to study their therapeutic potential, toxicity and pharmacokinetics. Due to the extreme genetic and phenotypic heterogeneity of OxPhos disorders one cannot rely on a single in ''vivo model''. Here we present the method and strategy we use to create, characterize and validate a set of ''Drosophila melanogaster'' models of nuclear DNA-encoded OxPhos subunits and preliminary results of systematic evaluation of Khondrion´s lead compound. We primarily focus on complex I by knocking down the core and accessory subunits the most prone to mutation in patients and selecting phenotypes-readouts suitable for drug screening (death at critical stages of development, survival curves, ROS level).These models will represent a valuable tool with predictive power to evaluate new potential therapeutics as an initial step in the drug development process.tial step in the drug development process.)
Kohoutova 2018 Physiol Res + (Ample experimental evidence suggests that … Ample experimental evidence suggests that sepsis could interfere with any mitochondrial function; however, the true role of mitochondrial dysfunction in the pathogenesis of sepsis-induced multiple organ dysfunction is still a matter of controversy. This review is primarily focused on mitochondrial oxygen consumption in various animal models of sepsis in relation to human disease and potential sources of variability in experimental results documenting decrease, increase or no change in mitochondrial respiration in various organs and species. To date, at least three possible explanations of sepsis-associated dysfunction of the mitochondrial respiratory system and consequently impaired energy production have been suggested: 1. Mitochondrial dysfunction is secondary to tissue hypoxia. 2. Mitochondria are challenged by various toxins or mediators of inflammation that impair oxygen utilization (cytopathic hypoxia). 3. Compromised mitochondrial respiration could be an active measure of survival strategy resembling stunning or hibernation. To reveal the true role of mitochondria in sepsis, sources of variability of experimental results based on animal species, models of sepsis, organs studied, or analytical approaches should be identified and minimized by the use of appropriate experimental models resembling human sepsis, wider use of larger animal species in preclinical studies, more detailed mapping of interspecies differences and organ-specific features of oxygen utilization in addition to use of complex and standardized protocols evaluating mitochondrial respiration.cols evaluating mitochondrial respiration.)
Greenway 2019 J Evol Biol + (Ample sperm production is essential for su … Ample sperm production is essential for successful male reproduction in many species. The amount of sperm a male can produce is typically constrained by the size of his testes, which can be energetically expensive to grow and maintain. Whilst the economics of ejaculate allocation has been the focus of much theoretical and empirical literature, relatively little attention has been paid to individual adult variation and plasticity at the source of sperm production, the testes themselves. We experimentally address this issue using the insect ''Narnia femorata'' Stål (Hemiptera: Coreidae). We established the metabolic cost of testicular tissue, then quantified variation in individual testes mass in response to multiple mate quality and quantity treatments. We uncovered extreme variation across individuals and considerable short-term effects of mating activity on testes dry mass. Importantly, the observed variation in testes mass was associated with notable fitness consequences; females paired with males with larger testes had greater hatching success. Overall, pairing with a female resulted in a 11% reduction in dry testes mass. Despite this apparent considerable mating investment, we found no evidence of strategic allocation to higher quality females or longer-term changes in testes mass. The dynamic nature of testes mass and its metabolic cost is vital to consider in the context of re-mating rates, polyandry benefits and general mating system dynamics both in this species and more broadly.© 2019 European Society For Evolutionary Biology. Journal of Evolutionary Biology © 2019 European Society For Evolutionary Biology.ropean Society For Evolutionary Biology.)
Miwa 2015 Free Radic Biol Med + (Amplex Red is a fluorescent probe that is … Amplex Red is a fluorescent probe that is widely used to detect hydrogen peroxide (H2O2) in a reaction where it is oxidised to resorufin by horseradish peroxidase (HRP) as a catalyst. This assay is highly rated amongst other similar probes thanks to its superior sensitivity and stability. However, we report here that Amplex Red is readily convertedto resorufin by a carboxylesterase without requiring H2O2, horseradish peroxidase or oxygen: this reaction is seen in various tissue samples such as liver and kidney as well as in cultured cells, causing a serious distortion of H2O2 measurements. The reaction can be inhibited by Phenylmethyl sulfonyl fluoride (PMSF) at concentrations which do not disturb mitochondrial function nor the ability of the Amplex Red-HRP system to detect H2O2. ''In vitro'' experiments and ''in silico'' docking simulations indicate that carboxylesterases 1 and 2 recognise Amplex Red with the same kinetics as carboxylesterase-containing mitochondria. We propose two different approaches to correct for this problem and re-evaluate the commonly performed experimental procedure for the detection of H2O2 release from isolated liver mitochondria. Our results call for a serious re-examination of previous data.on of H2O2 release from isolated liver mitochondria. Our results call for a serious re-examination of previous data.)
Dębski 2016 Free Radic Biol Med + (Amplex® Red (10-acetyl-3,7-dihydroxyphenox … Amplex® Red (10-acetyl-3,7-dihydroxyphenoxazine) is a fluorogenic probe widely used to detect and quantify hydrogen peroxide in biological systems. Detection of hydrogen peroxide is based on peroxidase-catalyzed oxidation of Amplex® Red to resorufin. In this study we investigated the mechanism of one-electron oxidation of Amplex® Red and we present the spectroscopic characterization of transient species formed upon the oxidation. Oxidation process has been studied by a pulse radiolysis technique with one-electron oxidants (N3(•), CO3(•-),(•)NO2 and GS(•)). The rate constants for the Amplex® Red oxidation by N3(•) ((2)k=2.1·10(9)M(-1)s(-1), at pH=7.2) and CO3(•-) ((2)k=7.6·10(8)M(-1)s(-1), at pH=10.3) were determined. Two intermediates formed during the conversion of Amplex® Red into resorufin have been characterized. Based on the results obtained, the mechanism of transformation of Amplex® Red into resorufin, involving disproportionation of the Amplex® Red-derived radical species, has been proposed. The results indicate that peroxynitrite-derived radicals, but not peroxynitrite itself, are capable to oxidize Amplex® Red to resorufin. We also demonstrate that horseradish peroxidase can catalyze oxidation of Amplex® Red not only by hydrogen peroxide, but also by peroxynitrite, which needs to be considered when employing the probe for hydrogen peroxide detection.the probe for hydrogen peroxide detection.)
Lopez 2017 Sci Rep + (Amyloid precursor protein (APP) and its ex … Amyloid precursor protein (APP) and its extracellular domain, soluble APP alpha (sAPPα) play important physiological and neuroprotective roles. However, rare forms of familial Alzheimer's disease are associated with mutations in APP that increase toxic amyloidogenic cleavage of APP and produce amyloid beta (Aβ) at the expense of sAPPα and other non-amyloidogenic fragments. Although mitochondrial dysfunction has become an established hallmark of neurotoxicity, the link between Aβ and mitochondrial function is unclear. In this study we investigated the effects of increased levels of neuronal APP or Aβ on mitochondrial metabolism and gene expression, in human SH-SY5Y neuroblastoma cells. Increased non-amyloidogenic processing of APP, but not Aβ, profoundly decreased respiration and enhanced glycolysis, while mitochondrial DNA (mtDNA) transcripts were decreased, without detrimental effects to cell growth. These effects cannot be ascribed to Aβ toxicity, since higher levels of endogenous Aβ in our models do not cause oxidative phosphorylation (OXPHOS) perturbations. Similarly, chemical inhibition of β-secretase decreased mitochondrial respiration, suggesting that non-amyloidogenic processing of APP may be responsible for mitochondrial changes. Our results have two important implications, the need for caution in the interpretation of mitochondrial perturbations in models where APP is overexpressed, and a potential role of sAPPα or other non-amyloid APP fragments as acute modulators of mitochondrial metabolism.te modulators of mitochondrial metabolism.)
Krako 2016 Abstract MitoFit Science Camp 2016 + (Amyloid β oligomers (AβOs) are crucially i … Amyloid β oligomers (AβOs) are crucially involved in Alzheimer’s disease (AD). They are still ˝mysterious entities˝ in terms of their precise molecular composition, their in-cell formation, traffic and actions. We have recently established a subcellularly localized conformational-selective interference approach, based on the expression of an intrabody against AβOs in the endoplasmic reticulum (ER) [1]. This intrabody is a recombinant antibody domain (scFvA13), previously isolated in our laboratory, that selectively binds to AβOs, with high selectivity and specificity with respect to Aβ monomeric species or fibrils [2]. For these studies, we exploited the 7PA2 cells (familial AD CHO cell model), the gold standard for natural AβOs production. Mitochondrial membrane potential was evaluated following the mitochondrial electrophoretic accumulation of the membrane permeable fluorescent cationic probe JC-1. Mitochondrial respiration was measured by high-resolution respirometry in intact and permeabilized cells. The concentration of ATP was quantified by chemiluminometry under basal cell metabolic conditions. Recently, we described that 7PA2 cells have another characteristic relevant to Alzheimer’s disease – a severe mitochondrial dysfunction, due to a chronic AβOs exposure and production [3]. We have newly established cell line, the 7PA2-A13K, as the model for conformational-selective interference with AβOs inside the endoplasmic reticulum (ER). These cells show a significantly lower level of secreted AβOs, without their intracellular accumulation and most importantly, through this modulatory effects on the levels and assembly of AβOs, a strong rescue of mitochondrial dysfunctions and bioenergetic deficit occurs (Figure 1). These data provide an important evidence for the fact that the AβOs are formed inside a cell, precisely in the ER, from where they exert their toxic action on mitochondrial physiology. These data contribute to the recent findings about the importance of the ER – mitochondria interplay in the pathogenesis of Alzheimer’s disease and highlight the subcellular compartment, called MAM – mitochondria associated ER membranes - as an important cellular target for the intracellular actions of AβOs. Future studies, that will extend these findings from a well defined cell model to neurons and, possibly to human neurons, will have to take into account the importance of organelle continuity and cross-talk for the cell homeostasis and AD pathology. From a methodological point of view, these results provide the first example of a functional experimental approach, conformational selective interference (CSI), that could find many applications for the studies of protein modifications ''in vivo''.dies of protein modifications ''in vivo''.)
Ng 2019 J Biol Chem + (Amyloid β1-42 (Aβ1-42) peptide is involved … Amyloid β1-42 (Aβ1-42) peptide is involved in Alzheimer's disease (AD) pathogenesis and is prone to glycation, an irreversible process resulting in proteins with accumulated advanced glycated end products (AGEs). Nε-(carboxyethyl)lysine (CEL) is a common AD-associated AGE, occurring at either Lys-16 or Lys-28 of Aβ1-42. Methylglyoxal is commonly used for the unspecific glycation of Aβ1-42, which results in a complex mixture of AGE-modified peptides and makes interpretation of a causative AGE at a specific amino acid residue difficult. Here, we addressed this challenge by chemically synthesizing defined CEL glycations on Aβ1-42 at Lys-16 (Aβ-CEL16), Lys-28 (Aβ-CEL28), and both Lys-16 and -28 (Aβ-CEL16&28). We demonstrate that the double CEL glycation at Lys-16/28 of Aβ1-42 had the most profound impact on amyloid fibril formation. ''In silico'' predictions indicated that Aβ-CEL16&28 had a substantially decreased free-energy change, contributing to fibril destabilization, and a decreased aggregation rate. Single CEL glycations at Lys-28 had the least impact on fibril formation, and Lys-16 CEL glycations delayed fibril formation. We also tested these peptides for neuronal toxicity and impact on mitochondrial function in a retinoic acid-differentiated SH-SY5Y human neuroblastoma cell line and found that only Aβ-CEL16 and Aβ-CEL28 are neurotoxic, possibly through a non-mitochondrial pathway, whereas Aβ-CEL16&28 is not neurotoxic. Interestingly, Aβ-CEL16&28 depolarized the mitochondrial membrane potential, and Aβ-CEL16 increased mitochondrial respiration at complex II, possibly indicating mitophagy or an alternative metabolic route, respectively. In summary, our results provide insights relevant for potential therapeutic approaches against neurotoxic CEL glycated Aβ1-42.Published under license by The American Society for Biochemistry and Molecular Biology, Inc.ety for Biochemistry and Molecular Biology, Inc.)
Souza da Silva 2020 Mol Neurobiol + (Amyloid-β oligomers (AβOs) toxicity causes … Amyloid-β oligomers (AβOs) toxicity causes mitochondrial dysfunction, leading to synaptic failure in Alzheimer's disease (AD). Considering presynaptic high energy demand and tight Ca2+ regulation, impairment of mitochondrial function can lead to deteriorated neural activity and cell death. In this study, an AD mouse model induced by ICV (intracerebroventricular) injection of AβOs was used to investigate the toxicity of AβOs on presynaptic function. As a therapeutic approach, GUO (guanosine) was given by oral route to evaluate the neuroprotective effects on this AD model. Following 24 h and 48 h from the model induction, behavioral tasks and biochemical analyses were performed, respectively. AβOs impaired object recognition (OR) short-term memory and reduced glutamate uptake and oxidation in the hippocampus. Moreover, AβOs decreased spare respiratory capacity, reduced ATP levels, impaired Ca2+ handling, and caused mitochondrial swelling in hippocampal synaptosomes. Guanosine crossed the BBB, recovered OR short-term memory, reestablished glutamate uptake, recovered mitochondrial Ca2+ homeostasis, and partially prevented mitochondrial swelling. Therefore, this endogenous purine presented a neuroprotective effect on presynaptic mitochondria and should be considered for further studies in AD models.nsidered for further studies in AD models.)
Cacabelos 2016 Acta Neuropathol Commun + (Amyotrophic lateral sclerosis (ALS) is a m … Amyotrophic lateral sclerosis (ALS) is a motor neuron disease with a gender bias towards major prevalence in male individuals. Several data suggest the involvement of oxidative stress and mitochondrial dysfunction in its pathogenesis, though differences between genders have not been evaluated. For this reason, we analysed features of mitochondrial oxidative metabolism, as well as mitochondrial chain complex enzyme activities and protein expression, lipid profile, and protein oxidative stress markers, in the Cu,Zn superoxide dismutase with the G93A mutation (hSOD1-G93A)- transgenic mice and Neuro2A(N2A) cells overexpressing hSOD1-G93A.Our results show that overexpression of hSOD1-G93A in transgenic mice decreased efficiency of mitochondrial oxidative phosphorylation, located at complex I, revealing a temporal delay in females with respect to males associated with a parallel increase in selected markers of protein oxidative damage. Further, females exhibit a fatty acid profile with higher levels of docosahexaenoic acid at 30 days. Mechanistic studies showed that hSOD1-G93A overexpression in N2A cells reduced complex I function, a defect prevented by 17β-estradiol pretreatment. In conclusion, ALS-associated SOD1 mutation leads to delayed mitochondrial dysfunction in female mice in comparison with males, in part attributable to the higher oestrogen levels of the former. This study is important in the effort to further understanding of whether different degrees of spinal cord mitochondrial dysfunction could be disease modifiers in ALS.unction could be disease modifiers in ALS.)
Magri 2022 Abstract Bioblast + (Amyotrophic lateral sclerosis (ALS) is a f … Amyotrophic lateral sclerosis (ALS) is a fatal neurodegenerative disease which affects motor neurons (MNs). Many familial and sporadic forms correlate with mutations in the gene encoding the antioxidant enzyme Cu/Zn Superoxide Dismutase (SOD1). Among mutants, the dismutase-active SOD1 G93A forms toxic aggregates on the cytosolic surface of outer mitochondrial membrane (OMM), using the Voltage-Dependent Anion Channel 1 (VDAC1) as binding site [1]. VDAC1 is the most abundant OMM pore-forming protein and allows the trafficking of metabolites (pyruvate, malate), ions, NAD+/NADH and ATP/ADP across the membrane; furthermore, it serves as an anchor for many cytosolic proteins, mostly for Hexokinases (HKs) [2]. However, in ALS MNs, the mitochondrial accumulation of SOD1 G93A impairs molecules exchange through VDAC1 and displaces HKs from mitochondria, promoting the organelle dysfunction and cell death [1-2].By a means of ''in vitro'' and ''in cellulo'' approaches, we previously demonstrated that HK1 and SOD1 G93A compete for the same mitochondrial binding site, VDAC1 [3]. Based on these observations, we developed a small synthetic peptide corresponding to the first 11 amino acid residues of the HK1 N-terminal domain (NHK1) [3]. NHK1 is able to modulates VDAC1 activity when it is reconstituted in artificial membranes; when added to ALS MNs, the peptide promotes a complete recovery of the cell viability in a dose-response manner [3-4]. By using High-Resolution Respirometry (HRR), we then analyzed the mitochondrial respiration profile of MN-like cells NSC34 stably expressing SOD1 G93A. Our results indicate that NHK1 promotes a partial increase of oxygen consumption corresponding to ROUTINE and OXPHOS state. As demonstrated by FCRs analysis, the peptide stimulates a significative decrease of the LEAK respiration while increases net respiration and coupling efficiency linked to OXPHOS state [4]. This effect is probably due to the reduction of ~70 % of VDAC1-SOD1 G93A aggregates observed in the mitochondrial fraction of cells treated with NHK1 [4].In conclusion, our results suggest that NHK1 drives the recovery of compromised mitochondrial respiration typical of ALS and provide new insights into the development of therapeutic molecules to fight the disease. Overall, our work helps to better understand the relationship between altered mitochondrial metabolism and MNs death.# Israelson A, Arbel N, da Cruz S, Ilieva H, Yamanaka K, Shoshan-Barmatz V, Cleveland DW (2010) Misfolded mutant SOD1 directly inhibits VDAC1 conductance in a mouse model of inherited ALS. https://doi.org/10.1016/j.neuron.2010.07.019# Magrì A, Reina S, De Pinto V (2018) VDAC1 as pharmacological target in cancer and neurodegeneration: focus on its role in apoptosis. https://doi.org/10.3389/fchem.2018.00108# Magrì A, Belfiore R, Reina S, Tomasello MF, Di Rosa MC, Guarino F, Leggio L, De Pinto V, Messina A (2016) Hexokinase I N-terminal based peptide prevents the VDAC1-SOD1 G93A interaction and re-establishes ALS cell viability. https://doi.org/10.1038/srep34802# Magrì A, Risiglione P, Caccamo A, Formicola B, Tomasello MF, Arrigoni C, Zimbone S, Guarino F, Re F, Messina A (2021) Small Hexokinase 1 peptide against toxic SOD1 G93A mitochondrial accumulation in ALS rescues the ATP-related respiration. https://doi.org/10.3390/biomedicines9080948ration. https://doi.org/10.3390/biomedicines9080948 )
Miquel 2014 Free Radic Biol Med + (Amyotrophic lateral sclerosis (ALS) is a f … Amyotrophic lateral sclerosis (ALS) is a fatal neurodegenerative disorder characterized by motor neuron degeneration that ultimately results in progressive paralysis and death. Growing evidence indicates that mitochondrial dysfunction and oxidative stress contribute to motor neuron degeneration in ALS. To further explore the hypothesis that mitochondrial dysfunction and nitroxidative stress contribute to disease pathogenesis at the ''in vivo'' level, we assessed whether the mitochondria-targeted antioxidant [10-(4,5-dimethoxy-2-methyl-3,6-dioxo-1,4- cyclohexadien-1-yl)decyl]triphenylphosphonium methanesulfonate (MitoQ) can modify disease progression in the SOD1G93A mouse model of ALS. To do this, we administered MitoQ (500µM) in the drinking water of SOD1G93A mice from a time when early symptoms of neurodegeneration become evident at 90 days of age until death. This regime is a clinically plausible scenario and could be more easily translated to patients as this corresponds to initiating treatment of patients after they are first diagnosed with ALS. MitoQ was detected in all tested tissues by liquid chromatography/mass spectrometry after 20 days of administration. MitoQ treatment slowed the decline of mitochondrial function, both in the spinal cord and quadriceps muscle as measured by high-resolution respirometry. Importantly, nitroxidative markers and pathological signs in the spinal cord of MitoQ-treated animals were markedly reduced and neuromuscular junctions were recovered associated to a significant increase in hind-limb strength. Finally, MitoQ treatment significantly prolonged the lifespan of SOD1G93A mice. Our results support a role for mitochondrial nitroxidative damage and dysfunction in the pathogenesis of ALS and suggest that mitochondria-targeted antioxidants may be of pharmacological use for ALS treatment. of pharmacological use for ALS treatment.)
Ladd 2017 Brain Res + (Amyotrophic lateral sclerosis (ALS) is a g … Amyotrophic lateral sclerosis (ALS) is a generally fatal neurodegenerative disease of adults that produces weakness and atrophy due to dysfunction and death of upper and lower motor neurons. We used RNA-sequencing (RNA-seq) to analyze expression of all mitochondrial DNA (mtDNA)-encoded respiratory genes in ALS and CTL human cervical spinal cords (hCSC) and isolated motor neurons. We analyzed with RNA-seq mtDNA gene expression in human neural stem cells (hNSC) exposed to recombinant human mitochondrial transcription factor A (rhTFAM), visualized in 3-dimensions clustered gene networks activated by rhTFAM, quantitated their interactions with other genes and determined their gene ontology (GO) families. RNA-seq and quantitative PCR (qPCR) analyses showed reduced mitochondrial gene expression in ALS hCSC and ALS motor neurons isolated by laser capture microdissection (LCM), and revealed that hNSC and CTL human cervical spinal cords were similar. Rats treated with i.v. rhTFAM showed a dose-response increase in brain respiration and an increase in spinal cord mitochondrial gene expression. Treatment of hNSC with rhTFAM increased expression of mtDNA-encoded respiratory genes and produced one major and several minor clusters of gene interactions. Gene ontology (GO) analysis of rhTFAM-stimulated gene clusters revealed enrichment in GO families involved in RNA and mRNA metabolism, suggesting mitochondrial-nuclear signaling. In postmortem ALS hCSC and LCM-isolated motor neurons we found reduced expression of mtDNA respiratory genes. In hNSC's rhTFAM increased mtDNA gene expression and stimulated mRNA metabolism by unclear mechanisms. rhTFAM may be useful in improving bioenergetic function in ALS.Copyright © 2017 Elsevier B.V. All rights reserved. © 2017 Elsevier B.V. All rights reserved.)
Martinez-Palma 2018 Neurotherapeutics + (Amyotrophic lateral sclerosis (ALS) is a f … Amyotrophic lateral sclerosis (ALS) is a fatal neurodegenerative disease characterized by motor neuron (MN) degeneration and gliosis. Neonatal astrocytes obtained from the SOD1G93A rat model of ALS exhibit mitochondrial dysfunction and neurotoxicity that can be reduced by dichloroacetate (DCA), a metabolic modulator that has been used in humans, and shows beneficial effects on disease outcome in SOD1G93A mice. Aberrant glial cells (AbGC) isolated from the spinal cords of adult paralytic SOD1G93A rats exhibit highly proliferative and neurotoxic properties and may contribute to disease progression. Here we analyze the mitochondrial activity of AbGC and whether metabolic modulation would modify their phenotypic profile. Our studies revealed fragmented mitochondria and lower respiratory control ratio in AbGC compared to neonatal SOD1G93A and nontransgenic rat astrocytes. DCA (5 mM) exposure improved AbGC mitochondrial function, reduced their proliferative rate, and importantly, decreased their toxicity to MNs. Furthermore, oral DCA administration (100 mg/kg, 10 days) to symptomatic SOD1G93A rats reduced MN degeneration, gliosis, and the number of GFAP/S100β double-labeled hypertrophic glial cells in the spinal cord. DCA treatment of AbGC reduced extracellular lactate levels indicating that the main recognized DCA action, targeting the pyruvate dehydrogenase kinase/pyruvate dehydrogenase complex, may underlie our findings. Our results show that AbGC metabolic phenotype is related to their toxicity to MNs and indicate that its modulation can reduce glial mediated pathology in the spinal cord. Together with previous findings, these results further support glial metabolic modulation as a valid therapeutic strategy in ALS.on as a valid therapeutic strategy in ALS.)
Veyrat-Durebex 2019 Mol Neurobiol + (Amyotrophic lateral sclerosis (ALS) is cha … Amyotrophic lateral sclerosis (ALS) is characterized by a wide metabolic remodeling, as shown by recent metabolomics and lipidomics studies performed in samples from patient cohorts and experimental animal models. Here, we explored the metabolome and lipidome of fibroblasts from sporadic ALS patients (n = 13) comparatively to age- and sex-matched controls (n = 11), and the subcellular fraction containing the mitochondria and endoplasmic reticulum (mito-ER), given that mitochondrial dysfunctions and ER stress are important features of ALS patho-mechanisms. We also assessed the mitochondrial oxidative respiration and the mitochondrial genomic (mtDNA) sequence, although without yielding significant differences. Compared to controls, ALS fibroblasts did not exhibit a mitochondrial respiration defect nor an increased proportion of mitochondrial DNA mutations. In addition, non-targeted metabolomics and lipidomics analyses identified 124 and 127 metabolites, and 328 and 220 lipids in whole cells and the mito-ER fractions, respectively, along with partial least-squares-discriminant analysis (PLS-DA) models being systematically highly predictive of the disease. The most discriminant metabolomic features were the alteration of purine, pyrimidine, and energetic metabolisms, suggestive of oxidative stress and of pro-inflammatory status. The most important lipidomic feature in the mito-ER fraction was the disturbance of phosphatidylcholine PC (36:4p) levels, which we had previously reported in the cerebrospinal fluid of ALS patients and in the brain from an ALS mouse model. Thus, our results reveal that fibroblasts from sporadic ALS patients share common metabolic remodeling, consistent with other metabolic studies performed in ALS, opening perspectives for further exploration in this cellular model in ALS.exploration in this cellular model in ALS.)
Tungtur 2021 Sci Rep + (Amyotrophic lateral sclerosis (ALS) remain … Amyotrophic lateral sclerosis (ALS) remains a devastating motor neuron disease with limited treatment options. Oxaloacetate treatment has a neuroprotective effect in rodent models of seizure and neurodegeneration. Therefore, we treated the ALS model superoxide dismutase 1 (SOD1) G93A mice with oxaloacetate and evaluated their neuromuscular function and lifespan. Treatment with oxaloacetate beginning in the presymptomatic stage significantly improved neuromuscular strength measured during the symptomatic stage in the injected mice compared to the non-treated group. Oxaloacetate treatment starting in the symptomatic stage significantly delayed limb paralysis compared with the non-treated group. For lifespan analysis, oxaloacetate treatment did not show a statistically significant positive effect, but the treatment did not shorten the lifespan. Mechanistically, SOD1YG93A mice showed increased levels of tumor necrosis factor-α (TNFα) and peroxisome proliferative activated receptor gamma coactivator 1α (PGC-1α) mRNAs in the spinal cord. However, oxaloacetate treatment reverted these abnormal levels to that of wild-type mice. Similarly, the altered expression level of total NF-κB protein returned to that of wild-type mice with oxaloacetate treatment. These results suggest that the beneficial effects of oxaloacetate treatment in SOD1G93A mice may reflect the effects on neuroinflammation or bioenergetic stress.> mice may reflect the effects on neuroinflammation or bioenergetic stress.)
Sauer 1979 Biochem J + (An NAD(P)+-dependent 'malic' enzyme is sho … An NAD(P)+-dependent 'malic' enzyme is shown to be present in mitochondria from small-intestinal mucosa. The intracellular location, activity and regulatory kinetic properties of the enzyme suggest that it participates in the major energy-producing pathway for net oxidation of glutamine-derived tricarboxylic acid-cycle intermediates.ed tricarboxylic acid-cycle intermediates.)
Nagel 1982 J Biol Chem + (An NAD(P)-dependent malic enzyme with a sp … An NAD(P)-dependent malic enzyme with a specific activity of 40.6 mumol of NADH/min/mg of protein and an isoelectric point of 5.4 was purified to apparent homogeneity from canine small intestinal mucosal mitochondria. The purification procedure employed ammonium sulfate fractionation, Sepharose CL 6B gel filtration, chromatography on DEAE-cellulose to remove the interfering malate dehydrogenase, and affinity chromatography on 2',5'-ADP-Sepharose and NAD-agarose to take advantage of the dual coenzyme specificity. Antibody prepared from the purified enzyme produced a single peak upon cross-rocket immunoelectrophoresis against the mitochondrial sonicate. Continuous polyacrylamide gel electrophoresis showed NAD and NADP activity co-migrating with the native protein band. A single band of protein having an apparent Mr = 62,000 was seen on sodium dodecyl sulfate electrophoresis. At pH 7.3, gel filtration revealed a single peak of activity with NAD and NADP corresponding to an apparent Mr = 282,000. Gradient gel polyacrylamide electrophoresis at pH 9.0 indicated an additional broad band of activity corresponding to a Mr = 141,000. Under physiological conditions therefore the protein appears to exist as a tetramer of Mr = 282,000 composed of four equal subunits, whereas at elevated pH values during electrophoresis, partial dissociation to a dimeric species occurs. dissociation to a dimeric species occurs.)
Marshall 1996 Am J Respir Cell Mol Biol + (An NADPH-oxidase complex containing at lea … An NADPH-oxidase complex containing at least two protein components (gp91-phox and p22-phox) and a unique low redox potential (-245 mV) cytochrome b-245 is the source of superoxide generated for bacterial killing in neutrophils and has been suggested as the oxygen sensor in the carotid body. In pure cultures of smooth muscle cells from calf small pulmonary arteries (300 microns diameter) the presence of the 91 kD protein specific to this cytochrome was demonstrated by Western blot analysis with monoclonal antibody 48. Low-temperature-difference spectrophotometry of homogenates of these cells demonstrated the characteristic cytochrome b-245 spectrum when titrated between redox potentials of -152 and -345 mV, consistent with the low redox potential form. When these same cells were exposed to hypoxia (approximately 40 mmHg), superoxide production increased significantly from 1.4 +/- 0.2 to 73 +/- 12 nmoles.min-1 mg-1 protein. Hypoxic generation of superoxide was inhibited by the NADPH-oxidase inhibitor diphenyleneiodonium (DPI: 10 microM) but not by the mitochondrial inhibitor myxathiazole (10 microM). The hypoxic superoxide increase was significantly greater than that observed from smooth muscle cells from large pulmonary arteries or from large or small systemic arteries. Fluorescence immunocytochemistry revealed the presence of the NADPH-oxidase protein in the walls of pulmonary arteries in rat lung slices, and confocal microscopy showed the complex to be widely distributed in the vicinity of the arterial smooth muscle walls. In hypoxia or norepinephrine (NP)-induced vasoconstriction of pulmonary artery rings from cats, the sensitivity to inhibition by DPI was observed to be significantly greater for hypoxia (ED50 = 0.8 microM) than for NP-induced (ED50 = 13.4 microM) constriction. Together these observations demonstrate that the unique cytochrome b-245 containing NADPH-oxidase is present in pulmonary artery smooth muscle and that an NADPH-oxidase or NADH-oxidoreductase complex is activated to release superoxide by hypoxic conditions. It is concluded that a trans-membrane NADPH-oxidase is the most likely and that activation of this system may be involved in the initiation of hypoxic pulmonary vasoconstriction.ion of hypoxic pulmonary vasoconstriction.)