Carnosic Acid Suppresses the H2O2-Induced Mitochondria-Related Bioenergetics Disturbances and Redox Impairment in SH-SY5Y Cells: Role for Nrf2.

The phenolic diterpene carnosic acid (CA, C20H28O4) exerts antioxidant, anti-inflammatory, anti-apoptotic, and anti-cancer effects in mammalian cells. CA activates the nuclear factor erythroid 2-related factor 2 (Nrf2), among other signaling pathways, and restores cell viability in several in vitro and in vivo experimental models. We have previously reported that CA affords mitochondrial protection against various chemical challenges. However, it was not clear yet whether CA would prevent chemically induced impairment of the tricarboxylic acid cycle (TCA) function in mammalian cells. In the present work, we found that a pretreatment of human neuroblastoma SH-SY5Y cells with CA at 1 µM for 12 h prevented the hydrogen peroxide (H2O2)-induced impairment of the TCA enzymes (aconitase, α-ketoglutarate dehydrogenase (α-KGDH), succinate dehydrogenase (SDH)) and abolished the inhibition of the complexes I and V and restored the levels of ATP by a mechanism associated with Nrf2. CA also exhibited antioxidant abilities by enhancing the levels of reduced glutathione (GSH) and decreasing the content oxidative stress markers (cellular 8-oxo-2'-deoxyguanosine (8-oxo-dG), and mitochondrial malondialdehyde (MDA), protein carbonyl, and 3-nitrotyrosine). Silencing of Nrf2 by small interfering RNA (siRNA) abrogated the protective effects elicited by CA in mitochondria of SH-SY5Y cells. Therefore, CA prevented the H2O2-triggered mitochondrial impairment by an Nrf2-dependent mechanism. The specific role of Nrf2 in ameliorating the function of TCA enzymes function needs further research.

Pinocembrin Suppresses H2O2-Induced Mitochondrial Dysfunction by a Mechanism Dependent on the Nrf2/HO-1 Axis in SH-SY5Y Cells.

Mitochondria are susceptible to redox impairment, which has been associated with neurodegeneration. These organelles are both a source and target of reactive species. In that context, there is increasing interest in finding natural compounds that modulate mitochondrial function and mitochondria-related signaling in order to prevent or to treat diseases involving mitochondrial impairment. Herein, we investigated whether and how pinocembrin (PB) would prevent mitochondrial dysfunction elicited by the exposure of human neuroblastoma SH-SY5Y cells to hydrogen peroxide (H2O2). PB (25 µM) was administrated for 4 h before H2O2 treatment (300 µM for 24 h). PB prevented H2O2-induced loss of cell viability mitochondrial depolarization in SH-SY5Y cells. PB also attenuated redox impairment in mitochondrial membranes. The production of superoxide anion radical (O2-•) and nitric oxide (NO•) was alleviated by PB in cells exposed to H2O2. PB suppressed the H2O2-induced inhibition of the tricarboxylic acid (TCA) cycle enzymes aconitase, α-ketoglutarate dehydrogenase, and succinate dehydrogenase. Furthermore, PB induced anti-inflammatory effects by abolishing the H2O2-dependent activation of the nuclear factor-κB (NF-κB) and upregulation of interleukin-1ß (IL-1ß) and tumor necrosis factor-α (TNF-α). The PB-induced antioxidant and anti-inflammatory effects are dependent on the heme oxygenate-1 (HO-1) enzyme and on the activation of the transcription factor nuclear factor erythroid 2-related factor 2 (Nrf2), since HO-1 inhibition (with 0.5 µM ZnPP IX) or Nrf2 silencing (with small interfering RNA (siRNA)) abolished the effects of PB. Overall, PB afforded cytoprotection by the Nrf2/HO-1 axis in H2O2-treated SH-SY5Y cells.

Tanshinone I Attenuates the Effects of a Challenge with H2O2 on the Functions of Tricarboxylic Acid Cycle and Respiratory Chain in SH-SY5Y Cells.

Tanshinone I (T-I; C18H12O3) is a cytoprotective molecule. T-I has been viewed as an antioxidant and anti-inflammatory agent exerting neuroprotective actions in several experimental models. Nonetheless, the mechanisms underlying the beneficial effects of T-I in mammalian cells are not completely understood yet. Mitochondrial dysfunction has been associated with several neurodegenerative diseases which remain uncured. Therefore, there is increasing interest in compounds that may be used in the prevention or treatment of those pathologies. Since T-I presents an antioxidant capacity, we investigated here whether and how this compound would prevent mitochondrial impairment in SH-SY5Y cells exposed to hydrogen peroxide (H2O2), which has been involved in the triggering of deleterious effects in several experimental models mimicking neurodegenerative processes. We found that a pretreatment with T-I at 2.5 µM for 2 h suppressed the pro-oxidant effects of H2O2 on mitochondrial membranes. Furthermore, T-I prevented the H2O2-elicited inhibition of the tricarboxylic acid (TCA) cycle enzymes (aconitase, α-ketoglutarate dehydrogenase, and succinate dehydrogenase) and of the mitochondrial complexes I and V. T-I also abrogated the mitochondrial depolarization and the mitochondrial failure to produce ATP in cells exposed to H2O2. T-I upregulated the levels of reduced glutathione (GSH) in the mitochondria of SH-SY5Y cells. T-I induced mitochondrial protection, at least in part, by activating the nuclear factor erythroid 2-related factor 2 (Nrf2), because silencing of Nrf2 by using small interference RNA (SiRNA) blocked these effects. Therefore, T-I afforded mitochondrial protection (involving both redox and bioenergetics-related aspects) against H2O2 through the activation of Nrf2.

Neurochemical evidence that glycine induces bioenergetical dysfunction.

Glycine tissue concentrations are increased particularly in nonketotic and ketotic hyperglycinemia, inherited metabolic disorders characterized by severe neurologic damage and brain abnormalities. The present work investigated the in vitro effects of glycine on important parameters of energy metabolism in the brain of young rats. The parameters analyzed were CO2 generated from glucose, acetate and citrate and the activities of the respiratory chain complexes I-IV, of the citric acid cycle enzymes citrate synthase, aconitase, isocitrate dehydrogenase, alpha-ketoglutarate dehydrogenase, succinate dehydrogenase, fumarase and malate dehydrogenase, of creatine kinase and Na+,K+-ATPase. Our results show that glycine significantly reduced CO2 production from acetate, but not from glucose and citrate, reflecting an impairment of the citric acid cycle function. We also observed that the activity of the mitochondrial enzyme citrate synthase was markedly inhibited by glycine, whereas the other activities of the citric acid cycle were not altered. Furthermore, the activity of the respiratory chain was reduced at complexes I-III, II-III and II, as well as of the mitochondrial isoform of creatine kinase and Na+,K+-ATPase. The data indicate that glycine severely impairs brain bioenergetics at the level of energy formation, transfer and utilization. Considering the importance of energy metabolism for brain development and functioning, it is presumed that glycine-induced impairment of brain energy homeostasis may be involved at least in part in the neurological damage found in patients affected by disorders in which brain glycine concentrations are increased.

Promotion of lipid and protein oxidative damage in rat brain by ethylmalonic acid.

High concentrations of ethylmalonic acid are found in tissues and biological fluids of patients affected by ethylmalonic encephalopathy, deficiency of short-chain acyl-CoA dehydrogenase activity and other illnesses characterized by developmental delay and neuromuscular symptoms. The pathophysiological mechanisms responsible for the brain damage in these patients are virtually unknown. Therefore, in the present work we investigated the in vitro effect of EMA on oxidative stress parameters in rat cerebral cortex. EMA significantly increased chemiluminescence and thiobarbituric acid-reactive species levels (lipoperoxidation), as well as carbonyl content and oxidation of sulfhydryl groups (protein oxidative damage) and DCFH. EMA also significantly decreased the levels of reduced glutathione (non-enzymatic antioxidant defenses). In contrast, nitrate and nitrite levels were not affected by this short organic acid. It is therefore presumed that oxidative stress may represent a pathomechanism involved in the pathophysiology of the neurologic symptoms manifested by patients affected by disorders in which EMA accumulates.

Evidence that 3-hydroxyisobutyric acid inhibits key enzymes of energy metabolism in cerebral cortex of young rats.

3-Hydroxyisobutyric aciduria is an inherited metabolic disease caused by 3-hydroxyisobutyryl-CoA dehydrogenase deficiency. Tissue accumulation and high urinary excretion of 3-hydroxyisobutyric acid is the biochemical hallmark of this disorder. Clinical phenotype is heterogeneous and generally includes dysmorphic features, delayed motor development, profound mental impairment, and acute encephalopathy. Lactic acidemia is also found in the affected patients, indicating that mitochondrial dysfunction may be involved in the pathophysiology of this disorder. Therefore, the aim of the present work was to investigate the in vitro effect of 3-hydroxyisobutyric acid (0.1, 0.5 and 1mM) on essential enzymes of energy metabolism, namely the activities of the respiratory chain complexes I-V, total, cytosolic and mitochondrial creatine kinase and Na(+), K(+)-ATPase in cerebral cortex homogenates of 30-day-old rats. We also measured the rate of oxygen consumption in brain mitochondrial preparations in the presence of 3-hydroxyisobutyric acid. 3-Hydroxyisobutyric acid significantly reduced complex I-III (20%), without affecting the other activities of the electron transport chain. Furthermore, 3-hydroxyisobutyric acid did not change state III, state IV and the respiratory control ratio in the presence of glutamate/malate or succinate, suggesting that its effect on cellular respiration was weak. On the other hand, the activities of total and mitochondrial creatine kinase, but not cytosolic creatine kinase, were inhibited (30%) by 3-hydroxyisobutyric acid. We also observed that 3-hydroxyisobutyric acid-induced inhibition of mitochondrial creatine kinase activity was fully prevented by pre-incubation of the homogenates with reduced glutathione, alpha-tocopherol or the combination of superoxide dismutase plus catalase, suggesting that this inhibition was mediated by oxidation of essential thiol groups of the enzyme probably by superoxide, hydrogen peroxide and/or peroxyl radicals. It was also demonstrated that Na(+), K(+)-ATPase activity from synaptic plasma membranes was markedly suppressed (37%) by 3-hydroxyisobutyric acid and that this effect was prevented by alpha-tocopherol co-incubation implying that peroxyl radicals were probably involved in this action. Considering the importance of the affected enzyme activities for brain metabolism homeostasis and neurotransmision, it is suggested that increased tissue levels of 3-hydroxyisobutyric acid may contribute to the neurodegeneration of patients affected by 3-hydroxyisobutyric aciduria and possibly explain previous reports describing elevated production and excretion of lactate.

Chronic early postnatal glutaric acid administration causes cognitive deficits in the water maze.

Glutaric acidemia type I (GA I) is an autosomal recessive metabolic disorder caused by glutaryl-CoA dehydrogenase deficiency leading to predominant accumulation of glutaric acid (GA), and to a lesser extent of 3-hydroxyglutaric acid (3HG) in body fluids and tissues. The clinical manifestations of GA I are predominantly neurological. Although the pathophysiological mechanisms responsible for the brain damage of this disease are virtually unknown, they are thought to be due to the neurotoxic actions of GA and 3HG. Therefore, in the present work we investigated whether chronic exposure of GA (5 micromol g of body weight(-1), twice per day), the major metabolite accumulating in GA I, during early development (from the 5th to the 28th day of life) could alter the cognitive performance of adult rats in the Morris water maze, open field and elevated plus maze tasks. Control rats were treated with saline in the same volumes. GA administration provoked an impairment of spatial performance in the water maze since adult rats pretreated with GA were not able to remember the previous location of the platform spending significantly less time in the training quadrant. In contrast, GA chronic administration did not affect rat performance in the open field and elevated plus maze tasks, indicating that motor activity and anxiety was not changed by GA. The results provide evidence that early chronic GA treatment induces long-lasting spatial behavioral deficit.

Kynurenines impair energy metabolism in rat cerebral cortex.

Growing evidence indicates that some metabolites derived from the kynurenine pathway, the major route of L-tryptophan catabolism, are involved in the neurotoxicity associated with several brain disorders, such as Huntington's disease, Parkinson's disease and Alzheimer's disease, as well as in glutaryl-CoA dehydrogenase deficiency (GAI). Considering that the pathophysiology of the brain damage in these neurodegenerative disorders is not completely defined, in the present study, we investigated the in vitro effect of L-kynurenine (Kyn), kynurenic acid (KA), 3-hydroxykynurenine (3HK), 3-hydroxyanthranilic acid (3HA) and anthranilic acid (AA) on some parameters of energy metabolism, namely glucose uptake, 14CO2 production from [U-14C] glucose, [1-14C] acetate and [1,5-14C] citrate, as well as on the activities of the respiratory chain complexes I-IV and Na+,K+-ATPase activity in cerebral cortex from 30-day-old rats. We observed that all compounds tested, except L-kynurenine, significantly increased glucose uptake and inhibited 14CO2 production from [U-14C] glucose, [1-14C] acetate and [1,5-14C] citrate. In addition, the activities of complexes I, II and IV of the respiratory chain were significantly inhibited by 3HK, while 3HA inhibited complexes I and II activities and AA inhibited complexes I-III activities. Moreover, Na+,K+-ATPase activity was not modified by these kynurenines. Taken together, our present data provide evidence that various kynurenine intermediates provoke impairment of brain energy metabolism.

In vitro effect of quinolinic acid on energy metabolism in brain of young rats.

Quinolinic acid (QA) is found at increased concentrations in brain of patients affected by various common neurodegenerative disorders, including Huntington's and Alzheimer's diseases. Considering that the neuropathology of these disorders has been recently attributed at least in part to energy deficit, in the present study we investigated the in vitro effect of QA (0.1-100 microM) on various parameters of energy metabolism, such as glucose uptake, (14)CO(2) production and lactate production, as well as on the activities of the respiratory chain complexes I-V, the citric acid cycle (CAC) enzymes, creatine kinase (CK), lactate dehydrogenase (LDH) and Na(+),K(+)-ATPase and finally the rate of oxygen consumption in brain of 30-day-old rats. We initially observed that QA significantly increased glucose uptake (55%), whereas (14)CO(2) generation from glucose, acetate and citrate was inhibited (up to 60%). Furthermore, QA-induced increase of brain glucose uptake was prevented by the NMDA receptor antagonist MK-801. Complex II activity was also inhibited (up to 35%) by QA, whereas the other activities of the respiratory chain complexes, CAC enzymes, CK and Na(+),K(+)-ATPase were not affected by the acid. Furthermore, inhibition of complex II activity was fully prevented by pre-incubating cortical homogenates with catalase plus superoxide dismutase, indicating that this effect was probably mediated by reactive oxygen species. In addition, lactate production was also not altered by QA, in contrast to the conversion of pyruvate to lactate catalyzed by LDH, which was significantly decreased (17%) by this neurotoxin. We also observed that QA did not change state III, state IV and the respiratory control ratio in the presence of glutamate/malate or succinate, suggesting that its effect on cellular respiration was rather weak. The data provide evidence that QA provokes a mild impairment of brain energy metabolism in vitro and does not support the view that the brain energy deficiency associated to certain neurodegenerative disorders could be solely endorsed to QA accumulation.

Inhibition of energy metabolism in cerebral cortex of young rats by the medium-chain fatty acids accumulating in MCAD deficiency.

Patients affected by medium-chain acyl CoA dehydrogenase (MCAD) deficiency, a frequent inborn error of metabolism, suffer from acute episodes of encephalopathy. However, the mechanisms underlying the neuropathology of this disease are poorly known. In the present study, we investigated the in vitro effect of the medium-chain fatty acids (MCFA), at concentrations varying from 0.01 to 3 mM, accumulating in MCAD deficiency on some parameters of energy metabolism in cerebral cortex of young rats. (14)CO(2) production from [U(14)] glucose, [1-(14)C] acetate and [1,5-(14)C] citrate was evaluated by incubating cerebral cortex homogenates from 30-day-old rats in the absence (controls) or presence of octanoic acid, decanoic acid or cis-4-decenoic acid. OA and DA significantly reduced (14)CO(2) production from acetate by around 30-40%, and from glucose by around 70%. DA significantly reduced (14)CO(2) production from citrate by around 40%, while OA did not affect this parameter. cDA inhibited (14)CO(2) production from all tested substrates by around 30-40%. The activities of the respiratory chain complexes and of creatine kinase were also tested in the presence of DA and cDA. Both metabolites significantly inhibited cytochrome c oxidase activity (by 30%) and complex II-III activity (DA, 25%; cDA, 80%). Furthermore, only cDA inhibited complex II activity (by 30%), while complex I-III and citrate synthase were not affected by these MCFA. On the other hand, only cDA reduced the activity of creatine kinase in total homogenates, as well as in mitochondrial and cytosolic fractions from cerebral cortex (by 50%). The data suggest that the major metabolites which accumulate in MCAD deficiency, with particular emphasis to cDA, compromise brain energy metabolism. We presume that these findings may contribute to the understanding of the pathophysiology of the neurological dysfunction of MCAD deficient patients.