Cleide Goncalves da Silva

Inhibition of the mitochondrial respiratory chain complex activities in rat cerebral cortex by methylmalonic acid

Propionic and methylmalonic acidemic patients have severe neurologic symptoms whose etiopathogeny is still obscure. Since increase of lactic acid is detected in the urine of these patients, especially during metabolic decompensation when high concentrations of methylmalonate (MMA) and propionate (PA) are produced, it is possible that cellular respiration may be impaired in these individuals. Therefore, we investigated the effects of MMA and PA (1, 2.5 and 5mM), the principal metabolites which accumulate in these conditions, on the mitochondrial respiratory chain complex activities succinate: 2,6-dichloroindophenol (DCIP) oxireductase (complex II); succinate: cytochrome c oxireductase (complexII+CoQ+III); NADH: cytochrome c oxireductase (complex I+CoQ+complex III); and cytochrome c oxidase (COX) (complex IV) from cerebral cortex homogenates of young rats. The effect of MMA on ubiquinol: cytochrome c oxireductase (complex III) and NADH: ubiquinone oxireductase (complex I) activities was also tested. Control groups did not contain MMA and PA in the incubation medium. MMA significantly inhibited complex I+III (32-46%), complex I (61-72%), and complex II+III (15-26%), without affecting significantly the activities of complexes II, III and IV. However, by using 1mM succinate in the assay instead of the usual 16mM concentration, MMA was able to significantly inhibit complex II activity in the brain homogenates. In contrast, PA did not affect any of these mitochondrial enzyme activities. The effect of MMA and PA on succinate: phenazine oxireductase (soluble succinate dehydrogenase (SDH)) was also measured in mitochondrial preparations. The results showed significant inhibition of the soluble SDH activity by MMA (11-27%) in purified mitochondrial fractions. Thus, if the in vitro inhibition of the oxidative phosphorylation system is also expressed under in vivo conditions, a deficit of brain energy production might explain some of the neurological abnormalities found in patients with methylmalonic acidemia (MMAemia) and be responsible for the lactic acidemia/aciduria identified in some of them.

Inhibition of cytochrome c oxidase activity in rat cerebral cortex and human skeletal muscle by D-2-hydroxyglutaric acid in vitro

L-2-Hydroxyglutaric (LGA) and D-2-hydroxyglutaric (DGA) acids are the characteristic metabolites accumulating in the neurometabolic disorders known as L-2-hydroxyglutaric aciduria and D-2-hydroxyglutaric aciduria, respectively. Although these disorders are predominantly characterized by severe neurological symptoms, the neurotoxic mechanisms of brain damage are virtually unknown. In this study we have evaluated the role of LGA and DGA at concentrations ranging from 0.01 to 5.0 mM on various parameters of energy metabolism in cerebral cortex slices and homogenates of 30-day-old Wistar rats, namely glucose uptake, CO(2) production and the respiratory chain enzyme activities of complexes I to IV. DGA significantly decreased glucose utilization (2.5 and 5.0 mM) by brain homogenates and CO(2) production (5 mM) by brain homogenates and slices, whereas LGA had no effect on either measurement. Furthermore, DGA significantly inhibited cytochrome c oxidase activity (complex IV) (EC 1.9.3.1) in a dose-dependent manner (35-95%) at doses as low as 0.5 mM, without compromising the other respiratory chain enzyme activities. In contrast, LGA did not interfere with these activities. Our results suggest that the strong inhibition of cytochrome c oxidase activity by increased levels of DGA could be related to the neurodegeneration of patients affected by D-2-hydroxyglutaric aciduria.

In vitro inhibition of Na+,K(+)-ATPase activity from rat cerebral cortex by guanidino compounds accumulating in hyperargininemia.

Hyperargininemia is a metabolic disorder biochemically characterized by tissue accumulation of arginine (Arg) and other guanidino compounds (GC). Convulsions, lethargy and psychomotor delay are predominant clinical features of this disease. Considering that some GC are epileptogenic and cause a decrease in membrane fluidity and that Na+,K(+)-ATPase, a membrane-bound enzyme, is essential for cellular excitability and is decreased in experimental and human epilepsy, in the present study we determined the in vitro effects of Arg, N-acetylarginine (NAA), argininic acid (AA) and homoarginine (HA) on the activity of Na+,K(+)-ATPase in the synaptic plasma membrane from cerebral cortex of young rats in the hope to identify a possible mechanism for the brain damage in hyperargininemia. The results showed that all GC tested, except Arg, significantly inhibited Na+,K(+)-ATPase activity at concentrations similar to those observed in plasma and CSF of patients with hyperargininemia. In addition, competition between NAA, AA and HA for the binding to the enzyme was observed, suggesting a common binding site for the GC. It is therefore possible that the inhibitory effect of GC on Na+,K(+)-ATPase may be related to the brain dysfunction observed in hyperargininemia.

Inhibition of creatine kinase activity from rat cerebral cortex by D-2-hydroxyglutaric acid in vitro.

D-2-Hydroxyglutaric acid (DGA) is the biochemical hallmark of patients affected by the neurometabolic disorder known as D-2-hydroxyglutaric aciduria (DHGA). Although this disease is predominantly characterized by severe neurological findings, the underlying mechanisms of brain injury are virtually unknown. In the present study, we investigated the effect of DGA on total, cytosolic, and mitochondrial creatine kinase (CK) activities from cerebral cortex of 30-day-old Wistar rats. Total CK activity (tCK) was measured in whole cell homogenates, whereas cytosolic and mitochondrial activities were measured in the cytosolic and mitochondrial preparations from cerebral cortex. We verified that CK activities were significantly inhibited by DGA (11-34% inhibition) at concentrations as low as 0.25 mM, being the mitochondrial fraction the most affected activity. Kinetic studies revealed that the inhibitory effect of DGA was non-competitive in relation to phosphocreatine. We also observed that this inhibition was fully prevented by pre-incubation of the homogenates with reduced glutathione, suggesting that the inhibitory effect of DGA on tCK activity is possibly mediated by oxidation of essential thiol groups of the enzyme. Considering the importance of CK activity for brain metabolism homeostasis, our results suggest that inhibition of this enzyme by increased levels of DGA may be related to the neurodegeneration of patients affected by DHGA.

Inhibition of Na+, K+-ATPase from rat brain cortex by propionic acid

BUFFERED propionic acid was injected s.c. into rats twice a day at 8 h intervals from the 6 to 21 days of age. Control rats received saline in the same volumes. The animals were weighed and killed by decapitation at 23 days. Whole brain and cerebral cortex were weighed and synaptic plasma membranes were prepared from cortex for the determination of Na+,K+-ATPase and Mg2+-ATPase activities. Body, whole brain and cortical weights were similar in the two groups, suggesting that propionic acid does not cause malnutrition in rats. Na+,K+-ATPase activity was significantly reduced by 30% in membranes from the propionate-treated group, whereas Mg2+-ATPase activity was not. In another set of experiments, synaptic plasma membranes were prepared from cerebral cortex of 23-day-old rats and incubated with propionic acid at final concentrations ranging from 0.1 to 2.0 mM. Na+,K+-ATPase activity, but not Mg2+-ATPase activity, was inhibited by 22–32%. Since propionic acid concentrations in plasma of chronically treated rats and of propionic acidaemic children are of the same order of magnitude as those tested in vitro, the results suggest that the inhibition of Na+,K+-ATPase activity may be related to the neurological dysfunction of patients affected by propionic acidaemia.

L-2-hydroxyglutaric acid inhibits mitochondrial creatine kinase activity from cerebellum of developing rats.

l‐2‐Hydroxyglutaric acid (LGA) is the biochemical hallmark of patients affected by the neurometabolic disorder known as l‐2‐hydroxyglutaric aciduria (LHGA). Although this disorder is predominantly characterized by severe neurological findings and pronounced cerebellum atrophy, the neurotoxic mechanisms of brain injury are virtually unknown. In the present study, we investigated the effect of LGA, at 0.25–5 mM concentrations, on total creatine kinase (tCK) activity from cerebellum, cerebral cortex, cardiac muscle and skeletal muscle homogenates of 30‐day‐old Wistar rats. CK activity was measured also in the cytosolic (Cy‐CK) and mitochondrial (Mi‐CK) fractions from cerebellum. We verified that tCK activity was significantly inhibited by LGA in the cerebellum, but not in cerebral cortex, cardiac muscle and skeletal muscle. Furthermore, CK activity from the mitochondrial fraction was inhibited by LGA, whereas that from the cytosolic fraction of cerebellum was not affected by the acid. Kinetic studies revealed that the inhibitory effect of LGA on Mi‐CK was non‐competitive in relation to phosphocreatine. Finally, we verified that the inhibitory effect of LGA on tCK was fully prevented by pre‐incubation of the homogenates with reduced glutathione (GSH), suggesting that this inhibition is possibly mediated by oxidation of essential thiol groups of the enzyme. Considering the importance of creatine kinase activity for energy homeostasis, our results suggest that the selective inhibition of this enzyme activity by increased levels of LGA could be possibly related to the cerebellar degeneration characteristically found in patients affected by l‐2‐hydroxyglutaric aciduria.

Inhibition of energy productionin vitro by glutaric acid in cerebral cortex of young rats

The present study investigated the effects of glutaric acid (GA), which predominantly accumulates in glutaric acidemia type I (GA-I), on somein vitro parameters of energy metabolism in cerebral cortex of rats. We first evaluated CO2 production from [U-14C] acetate, as well as ATP levels in brain of young Wistar rats. The effect of the acid on the activities of the respiratory chain complexes were also investigated. GA was tested at final concentrations ranging from 0.5 to 5.0 mM. GA significantly reduced brain CO2 production by 50% at the concentrations of 0.5 to 3.0 mM, ATP levels by 25% at the concentration of 3.0 mM, succinate:cytochrome C oxireductase (complex II plus CoQ plus complex III) by 25% at 5 mM concentration, and NADH:cytochrome C oxireductase (complex I plus CoQ plus complex III) by 25% at 2.5 and 5 mM concentrations. The results strongly indicate that GA impairs brain energy production. If these effects also occur in humans, it is possible that they may contribute to the neuropathology of patients affected by GA-I.

Alanine Prevents the Decrease of Na+,K+-ATPase Activity in Experimental Phenylketonuria

Our objective was to investigate the effect of alanine administration on Na+,K+-ATPase activity in cerebral cortex of rats subjected to chemically-induced phenylketonuria. Wistar rats were treated from the 6th to the 28th day of life with subcutaneous injections of either 2.6 μmol alanine or 5.2 μmol phenylalanine plus 2.6 μmol α-methylphenylalanine per g body weight or phenylalanine plus α-methylphenylalanine plus alanine in the same doses or equivalent volumes of 0.15 M saline. The animals were killed on the 29th or 60th day of life. Synaptic plasma membrane from cerebral cortex was prepared for Na+,K+-ATPase activity determination. The results showed that alanine injection prevents the decrease of Na+,K+-ATPase activity in animals subjected to experimental phenylketonuria. Therefore, in case the same effects are achieved with ingested alanine, it is possible that alanine supplementation may be an important dietary adjuvant for phenylketonuric patients.

L-Pyroglutamic Acid Inhibits Energy Production and Lipid Synthesis in Cerebral Cortex of Young Rats In Vitro

In the present study we investigated the effects of L-pyroglutamic acid (PGA), which predominantly accumulates in the inherited metabolic diseases glutathione synthetase deficiency (GSD) and γ-glutamylcysteine synthetase deficiency (GCSD), on some in vitro parameters of energy metabolism and lipid biosynthesis. We evaluated the rates of CO2 production and lipid synthesis from [U-14C]acetate, as well as ATP levels and the activities of creatine kinase and of the respiratory chain complexes I-IV in cerebral cortex of young rats in the presence of PGA at final concentrations ranging from 0.5 to 3 mM. PGA significantly reduced brain CO2 production by 50% at the concentrations of 0.5 to 3 mM, lipid biosynthesis by 20% at concentrations of 0.5 to 3 mM and ATP levels by 52% at the concentration of 3 mM. Regarding the enzyme activities, PGA significantly decreased NADH:cytochrome c oxireductase (complex I plus CoQ plus complex III) by 40% at concentrations of 0.5–3.0 mM and cytochrome c oxidase activity by 22–30% at the concentration of 3.0 mM, without affecting the activities of succinate dehydrogenase, succinate:DCPIP oxireductase (complex II), succinate:cytochrome c oxireductase (complex II plus CoQ plus complex III) or creatine kinase. The results strongly indicate that PGA impairs brain energy production. If these effects also occur in humans, it is possible that they may contribute to the neuropathology of patients affected by these diseases.

Neurodegeneration, Mitochondrial Dysfunction, and Oxidative Stress

Mitochondria are intracellular organelles that play a crucial role in energy metabolism. Most cell energy is obtained through mitochondrial metabolic pathways, especially the Krebs cycle and electron transport chain which is the main site for production of reactive oxygen species such as superoxide, hydrogen peroxide, and hydroxyl radicals. Brain tissue is highly sensitive to oxidative stress due to its high oxygen consumption, iron and lipid contents, and low activity of antioxidant defenses. Thus, energy metabolism impairment and oxidative stress are important events that have been related to the pathogenesis of diseases affecting the central nervous system. In the present issue, the pathogenesis of common Alzheimers (AD) and Parkinsons (PD) diseases is addressed in five papers. Mondragon-Rodriguez et al. proposed that phosphorylated tau protein could play the role of potential connector and that a combined therapy involving antioxidants and check points for synaptic plasticity during early stages of the disease could become a viable therapeutic option for AD treatment. This paper is accompanied by a study by T. Rohn that explores the potential role that the triggering receptor expressed on myeloid cells 2 (TREM2) normally plays and how loss of function may contribute to AD pathogenesis by enhancing oxidative stress and inflammation within the central nervous system. Additionally, O. Myhre et al. review the possible impact of environmental exposures in metal dyshomeostasis and inflammation in AD and PD. Furthermore, T. Omura et al. explore recent studies on the mechanism of endoplasmic reticulum stress-induced neuronal death related to PD, focusing on the involvement of human ubiquitin ligase HRD1 in the prevention of neuronal death as well as a potential therapeutic approach for PD based on the upregulation of HRD1. Lastly, S. Matsuda et al. showed a concise overview on the cellular functions of the mitochondrial kinase PINK1 and the relationship between Parkinsonism and mitochondrial dynamics, with particular emphasis on a mitochondrial damage response pathway and mitochondrial quality control. Three of the papers deal with aspects of oxidative stress implicated in the pathogenesis of neurodegenerative diseases. W. Liu et al. reviewed the current literature on the effects of oxidative stress due to exhaustive training on uncoupling protein 2 (UCP2) and Bcl-2/Bax in rat skeletal muscles. A.-M. Enciu et al. explore the possibility of oxidative-induced molecular mechanisms of blood-brain barrier disruption and tight junction protein expression alteration, in relation to aging and neurodegeneration. Moreover, M. Tajes et al. suggest that peroxynitrite induces cell death and is a very harmful agent in brain ischemia. A. Hosseini and M. Abdollahi review the pathogenesis of diabetic neuropathy with a focus on oxidative stress and introduced therapies dependent or independent of oxidative stress. A. Sekigawa et al. review the currently available evidence that neither mitochondria nor leucine-rich repeat kinase 2 (LRRK2) was present in the swellings of mice expressing P123H β-synuclein, suggesting that α- and β-synucleins might play differential roles in the mitochondrial pathology of α-synucleinopathies. The paper by P. F. Schuck et al. showed that trans-glutaconic acid is toxic to brain cells in vitro, by causing alterations in cell ion balance and probably neurotransmission, as well as oxidative stress in rat cerebral cortex. To complete the issue, M. J. Rodriguez et al. discuss the mitochondrial KATP channel as a new target to control microglia activity, avoid its toxic phenotype, and facilitate a positive disease outcome. Fortes et al. showed that 5TIO1 can protect the brain against neuronal damage regularly observed during neuropathologies. These papers are accompanied by a review by Z. Yu et al. on how the neuroglobins neuroprotection is related to mitochondria function and regulation. By compiling these papers, we hope to enrich our readers and researchers with respect to mitochondrial dysfunction, energy metabolism impairment, and oxidative stress in the pathophysiology of neurodegenerative diseases. Emilio L. Streck Grzegorz A. Czapski Cleide Goncalves da Silva