Non-synaptic Brain Mitochondria Research Articles

Unveiling a key role of oxaloacetate-glutamate interaction in regulation of respiration and ROS generation in nonsynaptic brain mitochondria using a kinetic model.

Glutamate plays diverse roles in neuronal cells, affecting cell energetics and reactive oxygen species (ROS) generation. These roles are especially vital for neuronal cells, which deal with high amounts of glutamate as a neurotransmitter. Our analysis explored neuronal glutamate implication in cellular energy metabolism and ROS generation, using a kinetic model that simulates electron transport details in respiratory complexes, linked ROS generation and metabolic reactions. The analysis focused on the fact that glutamate attenuates complex II inhibition by oxaloacetate, stimulating the latter’s transformation into aspartate. Such a mechanism of complex II activation by glutamate could cause almost complete reduction of ubiquinone and deficiency of oxidized form (Q), which closes the main stream of electron transport and opens a way to massive ROS generating transfer in complex III from semiquinone radicals to molecular oxygen. In this way, under low workload, glutamate triggers the respiratory chain (RC) into a different steady state characterized by high ROS generation rate. The observed stepwise dependence of ROS generation on glutamate concentration experimentally validated this prediction. However, glutamate’s attenuation of oxaloacetate’s inhibition accelerates electron transport under high workload. Glutamate-oxaloacetate interaction in complex II regulation underlies the observed effects of uncouplers and inhibitors and acceleration of Ca2+ uptake. Thus, this theoretical analysis uncovered the previously unknown roles of oxaloacetate as a regulator of ROS generation and glutamate as a modifier of this regulation. The model predicted that this mechanism of complex II activation by glutamate might be operative in situ and responsible for excitotoxicity. Spatial-time gradients of synthesized hydrogen peroxide concentration, calculated in the reaction-diffusion model with convection under a non-uniform local approximation of nervous tissue, have shown that overproduction of H2O2 in a cell causes excess of its level in neighbor cells.

Ischemic Preconditioning Protects Astrocytes against Oxygen Glucose Deprivation Via the Nuclear Erythroid 2-Related Factor 2 Pathway.

Induction of ischemic preconditioning (IPC) represents a potential therapy against cerebral ischemia by activation of adaptive pathways and modulation of mitochondria to induce ischemic tolerance to various cells and tissues. Mitochondrial dysfunction has been ascribed to contribute to numerous neurodegenerative conditions and cerebral ischemia. Nuclear erythroid 2-related factor 2 (Nrf2) is a transcription factor that has traditionally been involved in upregulating cellular antioxidant systems to combat oxidative stress in the brain; however, the association of Nrf2 with mitochondria in the brain remains unclear. In the present study, we investigated the effects of Nrf2 on (i) IPC-induced protection of astrocytes; (ii) OXPHOS protein expression; and (iii) mitochondrial supercomplex formation.Oxygen-glucose deprivation (OGD) was used as an in vitro model of cerebral ischemia and IPC in cultured rodent astrocytes derived from WT C57Bl/6J and Nrf2-/- mice. OXPHOS proteins were probed via western blotting, and supercomplexes were determined by blue native gel electrophoresis.IPC-induced cytoprotection in wild-type, but not Nrf2-/- mouse astrocyte cultures following a lethal duration of OGD. In addition, our results suggest that Nrf2 localizes to the outer membrane in non-synaptic brain mitochondria, and that a lack of Nrf2 in vivo produces altered supercomplex formation in mitochondria.Our findings support a role of Nrf2 in mediating IPC-induced protection in astrocytes, which can profoundly impact the ischemic tolerance of neurons. In addition, we provide novel evidence for the association of Nrf2 to brain mitochondria and supercomplex formation. These studies offer new targets and pathways of Nrf2, which may be heavily implicated following cerebral ischemia.

Glutamate induces H2O2 synthesis in nonsynaptic brain mitochondria

Mitochondrial reactive oxygen species regulate many important biological processes. We studied H2O2 formation by nonsynaptic brain mitochondria in response to the addition of low concentrations of glutamate, an excitatory neurotransmitter. We demonstrated that glutamate at concentrations from 10 to 50μM stimulated the H2O2 generation in mitochondria up to 4-fold, in a dose-dependent manner. The effect of glutamate was observed only in the presence of Ca2+ (20μM) in the incubation medium, and the rate of calcium uptake by the brain mitochondria was increased by up to 50% by glutamate. Glutamate-dependent effects were sensitive to the NMDA receptor inhibitors MK-801 (10μM) and D-AP5 (20μM) and the inhibitory neurotransmitter glycine (5mM). We have shown that the H2O2 formation caused by glutamate is associated with complex II and is dependent on the mitochondrial potential. We have found that nonsynaptic brain mitochondria are a target of direct glutamate signaling, which can specifically activate H2O2 formation through mitochondrial respiratory chain complex II. The H2O2 formation induced by glutamate can be blocked by glycine, an inhibitory neurotransmitter that prevents the deleterious effects of glutamate in brain mitochondria.

Tacrine and its analogues impair mitochondrial function and bioenergetics: a lipidomic analysis in rat brain

Tacrine is an acetylcholinesterase (AChE) inhibitor used as a cognitive enhancer in the treatment of Alzheimer's disease (AD). However, its low therapeutic efficiency and a high incidence of side effects have limited its clinical use. In this study, the molecular mechanisms underlying the impact on brain activity of tacrine and two novel tacrine analogues (T1, T2) were approached by focusing on three aspects: (i) their effects on brain cholinesterase activity; (ii) perturbations on electron transport chain enzymes activities of non-synaptic brain mitochondria; and (iii) the role of mitochondrial lipidome changes induced by these compounds on mitochondrial bioenergetics. Brain effects were evaluated 18 h after the administration of a single dose (75.6 μmol/kg) of tacrine or tacrine analogues. The three compounds promoted a significant reduction in brain AChE and butyrylcholinesterase (BuChE) activities. Additionally, tacrine was shown to be more efficient in brain AChE inhibition than T2 tacrine analogue and less active than T1 tacrine analogue, whereas BuChE inhibition followed the order: T1 > T2 > tacrine. The studies using non-synaptic brain mitochondria show that all the compounds studied disturbed brain mitochondrial bioenergetics mainly via the inhibition of complex I activity. Furthermore, the activity of complex IV is also affected by tacrine and T1 treatments while FoF(1) -ATPase is only affected by tacrine. Therefore, the compounds' toxicity as regards brain mitochondria, which follows the order: tacrine >> T1 > T2, does not correlate with their ability to inhibit brain cholinesterase enzymes. Lipidomics approaches show that phosphatidylethanolamine (PE) is the most abundant phospholipids (PL) class in non-synaptic brain mitochondria and cardiolipin (CL) present the greatest diversity of molecular species. Tacrine induced significant perturbations in the mitochondrial PL profile, which were detected by means of changes in the relative abundance of phosphatidylcholine (PC), PE, phosphatidylinositol (PI) and CL and by the presence of oxidized phosphatidylserines. Additionally, in both the T1 and T2 groups, the lipid content and molecular composition of brain mitochondria PL are perturbed to a lesser extent than in the tacrine group. Abnormalities in CL content and the amount of oxidized phosphatidylserines were associated with significant reductions in mitochondrial enzymes activities, mainly complex I. These results indicate that tacrine and its analogues impair mitochondrial function and bioenergetics, thus compromising the activity of brain cells.

Acute ammonia neurotoxicity in vivo involves increase in cytoplasmic protein P53 without alterations in other markers of apoptosis

Acute intoxication with large ammonia doses leads to activation of NMDA receptors in the brain, resulting in oxidative stress and disturbance of mitochondrial function. Altered mitochondrial function is a crucial step in some mechanisms of cellular apoptosis. This study assesses whether ammonia intoxication in vivo leads to induction of apoptotic markers such as permeability transition pore (PTP) formation, caspase-3, and caspase-9 activation, changes in p53 protein, or cytochrome c release. Acute ammonia intoxication did not affect caspase-9 or caspase-3 activities. The mitochondrial membrane potential also remained unaltered in non-synaptic brain mitochondria after injection of ammonia, indicating that ammonia did not induce PTP formation in brain in vivo. The nuclear level of p53 did not change, whereas its cytoplasmic level increased approximately two-fold. In agreement with the theory that translocation of the p53 from cytosol to nuclei is an essential step for induction of apoptosis we did not find apoptotic nuclei in brain of rats injected with ammonia. This supports the idea that ammonia neurotoxicity does not involve apoptosis and points to impaired p53 transfer from cytoplasm to nuclei as a possible preventer of apoptosis. We did not find any release of cytochrome c from mitochondria to cytosol after ammonia injection. Cytochrome c content was significantly reduced (30%) in brain mitochondria from rats injected with ammonia. This decrease may contribute to the reduced state 3 respiration, decreased respiratory control index, and disturbances in the mitochondrial electron transport chain in brain mitochondria from rats injected with ammonia.

Lithium Desensitizes Brain Mitochondria to Calcium, Antagonizes Permeability Transition, and Diminishes Cytochrome c Release

Among the numerous effects of lithium on intracellular targets, its possible action on mitochondria remains poorly explored. In the experiments with suspension of isolated brain mitochondria, replacement of KCl by LiCl suppressed mitochondrial swelling, depolarization, and a release of cytochrome c induced by a single Ca2+ bolus. Li+ robustly protected individual brain mitochondria loaded with rhodamine 123 against Ca2+-induced depolarization. In the experiments with slow calcium infusion, replacement of KCl by LiCl in the incubation medium increased resilience of synaptic and nonsynaptic brain mitochondria as well as resilience of liver and heart mitochondria to the deleterious effect of Ca2+. In LiCl medium, mitochondria accumulated larger amounts of Ca2+ before they lost the ability to sequester Ca2+. However, lithium appeared to be ineffective if mitochondria were challenged by Sr2+ instead of Ca2+. Cyclosporin A, sanglifehrin A, and Mg2+, inhibitors of the mitochondrial permeability transition (mPT), increased mitochondrial Ca2+ capacity in KCl medium but failed to do so in LiCl medium. This suggests that the mPT might be a common target for Li+ and mPT inhibitors. In addition, lithium protected mitochondria against high Ca2+ in the presence of ATP, where cyclosporin A was reported to be ineffective. SB216763 and SB415286, inhibitors of glycogen synthase kinase-3beta, which is implicated in regulating reactive oxygen species-induced mPT in cardiac mitochondria, did not increase Ca2+ capacity of brain mitochondria. Altogether, these findings suggest that Li+ desensitizes mitochondria to elevated Ca2+ and diminishes cytochrome c release from brain mitochondria by antagonizing the Ca2+-induced mPT.

Apoptotic markers in the mitochondria, cytosol, and nuclei of brain cells during ammonia toxicity

The effects of toxic doses of ammonia on the induction of pro-apoptotic markers in rat brain cells were studied. It was found that neither induction of caspase-9 and caspase-3, nor changes in the membrane potential in nonsynaptic brain mitochondria, release of cytochrome c from mitochondria to cytosol, or apoptosis-inducing signals in the cell nuclei, were responsible for the ammonia toxicity in vivo. Thus, no evidence was found for any functional significance of mitochondria in cell death associated with acute hyperammonemia. The resistance of cell nuclei to ammonia-induced apoptosis could be caused either by a disordered translocation of the p53 protein from the cytosol into the nucleus or by translocation of its inactive form. The nonsynaptic brain mitochondria were also shown to be more resistant to the formation of nonspecific permeability pores and to Ca2+-induced swelling than cardiac and hepatic mitochondria and, as well, supported their membrane potential even under conditions of ammonia-stimulated production of active oxygen metabolites, excessive accumulation of Ca2+, oxidative stress, and impaired energy metabolism.

Sources of oxygen radicals in brain in acute ammonia intoxication in vivo

The effects of acute ammonia intoxication on reactive oxygen species production by different sources in rat brain were studied. Ammonia intoxication in vivo leads to reduced activity of superoxide dismutase (SOD), catalase and glutathione peroxidase in brain nonsynaptic mitochondria and increased formation of O 2 − by submitochondrial particles. It also results in increased xanthine oxidase (XO) activity and decreased xanthine dehydrogenase (XDH)/XO activity ratio indicating conversion of XDH to XO and also increases monoamine oxidase A (MAO-A) activity but not of MAO-B. Blocking NMDA receptors with MK-801 prevents ammonia-induced oxidative stress, XDH to XO conversion and MAO-A activation. Ammonia intoxication did not lead to H 2O 2 formation by mitochondria, in spite of increased O 2 − generation. The main source of H 2O 2 in the mitochondrial matrix was Mn-SOD. Ammonia intoxication in vivo leads to increased superoxide and decreased hydrogen peroxide in nonsynaptic brain mitochondria. Increased superoxide is due to increased formation by the respiratory chain and by xanthine and aldehyde oxidases and decreased elimination by antioxidant enzymes. The reduced formation of hydrogen peroxide is due to the reduced activity of Mn-SOD. Prevention of ammonia-induced production of reactive oxygen species by MK-801 supports the idea that it is mediated by activation of NMDA receptors.

Calcium and ammonia stimulate monoamine oxidase A activity in brain mitochondria

The effect of ammonia and calcium on the activity of monoamine oxidase (MAO) was studied. The enzyme activity in non-synaptic brain mitochondria isolated from rats administered with ammonium acetate was assayed by release of H2O2 using spectrophotometry. The effect of calcium on MAO was assayed directly after addition of Ca2+ to the non-synaptic mitochondria isolated from the forebrain of the control rats. Both ammonium acetate injection in vivo and Ca2+ addition in vitro stimulated the activity of MAO A but not that of MAO B in mitochondria. This is the first evidence that ammonia and Ca2+ regulate MAO A in non-synaptic mitochondria in the forebrain and can contribute to oxidative stress in the neurons via MAO A activation.

Mitochondrial oxygen radical generation and leak: sites of production in states 4 and 3, organ specificity, and relation to aging and longevity.

Studies in heart and nonsynaptic brain mitochondria from two mammals and three birds show that complex I generates oxygen radicals in heart and nonsynaptic brain mitochondria in States 4 and 3, whereas complex III does it only in heart mitochondria and only in State 4. The increase in oxygen consumption during the State 4 to 3 transition is not accompanied by a proportional increase in oxygen radical generation. This will protect mitochondria and tissues during bursts of activity. Comparisons between young and old rodents do not show a consistent pattern of variation in mitochondrial oxygen radical production during aging. However, all the interspecies comparisons performed to date between different mammals, and between mammals and birds, agree that animals with high maximum longevities have low rates of mitochondrial oxygen radical production, irrespective of the value of their basal specific metabolic rate. The sites and mechanisms allowing this, the recently described low degree of membrane fatty acid unsaturation of longevous animals, and their relation to longevity and aging are discussed.

Mitochondrial free radical production and aging in mammals and birds.

The mitochondrial rate of oxygen radical (ROS) production is negatively correlated with maximum life span potential (MLSP) in mammals following the rate of living theory. In order to know if this relationship is more than circumstantial, homeothermic vertebrates with MLSP different from that predicted by the body size and metabolic rate of the majority of mammals (like birds and primates) must be studied. Birds are unique because they combine a high rate of basal oxygen consumption with a high MLSP. Heart, brain, and lung mitochondrial ROS production and free radical leak (percent of total electron flow directed to ROS production) are lower in three species of birds of different orders than in mammals of similar body size and metabolic rate. This suggests that the capacity to show a low rate of ROS production is a general characteristic of birds. Using substrates and inhibitors specific for different segments of the respiratory chain, the main ROS generator site (responsible for those bird-mammalian differences) in state 4 has been localized at complexes I and III in heart mitochondria and only at complex I in nonsynaptic brain mitochondria. In state 3, complex I is the only generator in both tissues. The results also suggest that the iron-sulphur centers are the ROS generators of complex I. A general mechanism that allows pigeon mitochondria to show a low rate of ROS production can be the capacity to maintain a low degree of reduction of the ROS generator site. In heart mitochondria, this is supplemented with a low rate of oxygen consumption physiologically compensated with a comparatively higher heart size. A low rate of free radical production near DNA, together with a high rate of DNA repair, can be responsible for the slow rate of accumulation of DNA damage and thus the slow aging rate of longevous animals.

Localization at complex I and mechanism of the higher free radical production of brain nonsynaptic mitochondria in the short-lived rat than in the longevous pigeon.

Free radical production and leak of brain nonsynaptic mitochondria were higher with pyruvate/malate than with succinate in rats and pigeons. Rotenone, antimycin A, and myxothiazol maximally stimulated free radical production with pyruvate/malate but not with succinate. Simultaneous treatment with myxothiazol plus antimycin A did not decrease the stimulated rate of free radical production brought about independently by any of these two inhibitors with pyruvate/malate. Thenoyltrifluoroacetone did not increase free radical production with succinate. No free radical production was detected at Complex IV. Free radical production and leak with pyruvate/malate were higher in the rat (maximum longevity 4 years) than in the pigeon (maximum longevity 35 years). These differences between species disappeared in the presence of rotenone. The results localize the main free radical production site of nonsynaptic brain mitochondria at Complex I. They also suggest that the low free radical production of pigeon brain mitochondria is due to a low degree of reduction of Complex I in the steady state in this highly longevous species.

Effect of age on kinetics and carbon monoxide binding to cytochrome oxidase in synaptic and non-synaptic brain mitochondria

The kinetic parameters of cytochrome oxidase activity in synaptic and non-synaptic brain mitochondria from 3- and 30-month-old rats were determined at room temperature. The value of K m for cytochrome c increased only 12–13% with age. The maximal velocity did not change with age, but the value of V max in synaptic mitochondria is twice that observed in non-synaptic mitochondrial cytochrome oxidase. The kinetics of CO binding to cytochrome oxidase at temperatures from 183 to 225K were also studied in synaptic and non-synaptic mitochondria from 3- and 30-month-old rat forebrains. Age-dependent differences were observed only in mitochondria of synaptic origin. Following flash photolysis at low temperatures, CO migration to the iron requires crossing two free energy barriers separating two intermediate regions from the iron. In 3-month-old synaptic mitochondria, CO must migrate across a 10.3 kca/mol barrier separating two intermedite regions, I 2 and I; a 4.7 kcal/mol barrier separates the innermost region I from the iron. Each intermediate region in 3-month-old cytochrome oxidase can hold only one CO molecule. In 30-month-old synaptic mitochondria, 10.3 kcal/mol barriers separate the two intermediate regions as well as region I and the iron; each intermediate region can hold two CO molecules. Region I 2 in non-synaptic cytochrome oxidase at either age can hold two CO molecules and the innermost region I holds only one CO molecule; energy barriers of approximately 10.3 kcal/moIe separate regions I 2, I, and the iron. These age-dependent changes may reflect age-dependent conformational changes in cytochrome oxidase.

Spectrophotometry of b-type cytochromes in rat brain in vivo and in vitro

Terminal oxidase inhibitors such as cyanide (CN) and carbon monoxide (CO) produce different absorption changes in the intact brain, suggesting different mitochondrial responses to the inhibitors. In the present study, the nature of the cytochromes involved in CO and CN responses in vivo was investigated by low-temperature spectroscopy of rat brain, frozen in situ, and of preparations of brain homogenate and isolated mitochondria. Comparison of the spectra from different preparations at the high resolution afforded by low-temperature spectroscopy indicated that absorption responses to CO in vivo originated from mitochondrial b cytochromes. Further detailed spectral analysis of mitochondrial preparations revealed three CN-insensitive b cytochromes in nonsynaptic brain mitochondria; one cytochrome could be reduced by succinate in the presence of CN, the second could be reduced by succinate plus ATP, and the third could be reduced only by anaerobiosis. The spectral characteristics of the mitochondrial b cytochromes, when compared with spectra from CO-exposed brain tissue frozen in situ, strongly implicated the energy-dependent cytochrome b in the oxidation-reduction (redox) responses caused by CO in vivo.