Evaluation of the effects of the Zika Virus-Immunoglobulin G+ complex on murine microglial cells.

After the Zika virus (ZIKV) epidemic in Brazil, ZIKV infections were linked to damage to the central nervous system (CNS) and congenital anomalies. Due to the virus's ability to cross the placenta and reach brain tissue, its effects become severe, leading to Congenital Zika Syndrome (CZS) and resulting in neuroinflammation, microglial activation, and secretion of neurotoxic factors. The presence of ZIKV triggers an inadequate fetal immune response, as the fetus only has the protection of maternal antibodies of the Immunoglobulin G (IgG) class, which are the only antibodies capable of crossing the placenta. Because of limited understanding regarding the long term consequences of ZIKV infection and the involvement of maternal antibodies, this study sought to assess the impact of the ZIKV + IgG⁺complex on murine microglial cells. The cells were exposed to ZIKV, IgG antibodies, and the ZIKV + IgG⁺complex for 24 and 72 h. Treatment-induced cytotoxic effects were evaluated using the cell viability assay, oxidative stress, and mitochondrial membrane potential. The findings indicated that IgG antibodies exhibit cytotoxic effects on microglia, whether alone or in the presence of ZIKV, leading to compromised cell viability, disrupted mitochondrial membrane potential, and heightened oxidative damage. Our conclusion is that IgG antibodies exert detrimental effects on microglia, triggering their activation and potentially disrupting the creation of a neurotoxic environment. Moreover, the presence of antibodies may correlate with an elevated risk of ZIKV-induced neuroinflammation, contributing to long-term CNS damage.

S-adenosylmethionine induces mitochondrial dysfunction, permeability transition pore opening and redox imbalance in subcellular preparations of rat liver.

S-adenosylmethionine (AdoMet) predominantly accumulates in tissues and biological fluids of patients affected by liver dysmethylating diseases, particularly glycine N-methyltransferase, S-adenosylhomocysteine hydrolase and adenosine kinase deficiencies, as well as in some hepatic mtDNA depletion syndromes, whose pathogenesis of liver dysfunction is still poorly established. Therefore, in the present work, we investigated the effects of S-adenosylmethionine (AdoMet) on mitochondrial functions and redox homeostasis in rat liver. AdoMet decreased mitochondrial membrane potential and Ca2+ retention capacity, and these effects were fully prevented by cyclosporin A and ADP, indicating mitochondrial permeability transition (mPT) induction. It was also verified that the thiol-alkylating agent NEM prevented AdoMet-induced ΔΨm dissipation, implying a role for thiol oxidation in the mPT pore opening. AdoMet also increased ROS production and provoked protein and lipid oxidation. Furthermore, AdoMet reduced GSH levels and the activities of aconitase and α-ketoglutarate dehydrogenase. Free radical scavengers attenuated AdoMet effects on lipid peroxidation and GSH levels, supporting a role of ROS in these effects. It is therefore presumed that disturbance of mitochondrial functions associated with mPT and redox unbalance may represent relevant pathomechanisms of liver damage provoked by AdoMet in disorders in which this metabolite accumulates.

Ornithine and Homocitrulline Impair Mitochondrial Function, Decrease Antioxidant Defenses and Induce Cell Death in Menadione-Stressed Rat Cortical Astrocytes: Potential Mechanisms of Neurological Dysfunction in HHH Syndrome.

Hyperornithinemia-hyperammonemia-homocitrullinuria (HHH) syndrome is caused by deficiency of ornithine translocase leading to predominant tissue accumulation and high urinary excretion of ornithine (Orn), homocitrulline (Hcit) and ammonia. Although affected patients commonly present neurological dysfunction manifested by cognitive deficit, spastic paraplegia, pyramidal and extrapyramidal signs, stroke-like episodes, hypotonia and ataxia, its pathogenesis is still poorly known. Although astrocytes are necessary for neuronal protection. Therefore, in the present study we investigated the effects of Orn and Hcit on cell viability (propidium iodide incorporation), mitochondrial function (thiazolyl blue tetrazolium bromide-MTT-reduction and mitochondrial membrane potential-ΔΨm), antioxidant defenses (GSH) and pro-inflammatory response (NFkB, IL-1ß, IL-6 and TNF-α) in unstimulated and menadione-stressed cortical astrocytes that were previously shown to be susceptible to damage by neurotoxins. We first observed that Orn decreased MTT reduction, whereas both amino acids decreased GSH levels, without altering cell viability and the pro-inflammatory factors in unstimulated astrocytes. Furthermore, Orn and Hcit decreased cell viability and ΔΨm in menadione-treated astrocytes. The present data indicate that the major compounds accumulating in HHH syndrome impair mitochondrial function and reduce cell viability and the antioxidant defenses in cultured astrocytes especially when stressed by menadione. It is presumed that these mechanisms may be involved in the neuropathology of this disease.

Ornithine In Vivo Administration Disrupts Redox Homeostasis and Decreases Synaptic Na(+), K (+)-ATPase Activity in Cerebellum of Adolescent Rats: Implications for the Pathogenesis of Hyperornithinemia-Hyperammonemia-Homocitrullinuria (HHH) Syndrome.

Hyperornithinemia-hyperammonemia-homocitrullinuria (HHH) syndrome is an inborn error of metabolism caused by a defect in the transport of ornithine (Orn) into mitochondrial matrix leading to accumulation of Orn, homocitrulline (Hcit), and ammonia. Affected patients present a variable clinical symptomatology, frequently associated with cerebellar symptoms whose pathogenesis is poorly known. Although in vitro studies reported induction of oxidative stress by the metabolites accumulating in HHH syndrome, so far no report evaluated the in vivo effects of these compounds on redox homeostasis in cerebellum. Therefore, the present work was carried out to investigate the in vivo effects of intracerebellar administration of Orn and Hcit on antioxidant defenses (reduced glutathione concentrations and the activities of superoxide dismutase, catalase, glutathione peroxidase, glutathione reductase, and glucose-6-phosphate dehydrogenase), lipid oxidation (malondialdehyde concentrations), as well as on the activity of synaptic Na(+), K(+)-ATPase, an enzyme highly vulnerable to free radical attack, in the cerebellum of adolescent rats. Orn significantly increased malondialdehyde levels and the activities of all antioxidant enzymes, and reduced Na(+), K(+)-ATPase activity. In contrast, glutathione concentrations were not changed by Orn treatment. Furthermore, intracerebellar administration of Hcit was not able to alter any of these parameters. The present data show for the first time that Orn provokes in vivo lipid oxidative damage, activation of the enzymatic antioxidant defense system, and reduction of the activity of a crucial enzyme involved in neurotransmission. It is presumed that these pathomechanisms may contribute at least partly to explain the neuropathology of cerebellum abnormalities and the ataxia observed in patients with HHH syndrome.

Pristanic acid provokes lipid, protein, and DNA oxidative damage and reduces the antioxidant defenses in cerebellum of young rats.

Zellweger syndrome (ZS) and some peroxisomal diseases are severe inherited disorders mainly characterized by neurological symptoms and cerebellum abnormalities, whose pathogenesis is poorly understood. Biochemically, these diseases are mainly characterized by accumulation of pristanic acid (Prist) and other fatty acids in the brain and other tissues. In this work, we evaluated the in vitro influence of Prist on redox homeostasis by measuring lipid, protein, and DNA damage, as well as the antioxidant defenses and the activities of aconitase and α-ketoglutarate dehydrogenase in cerebellum of 30-day-old rats. The effect of Prist on DNA damage was also evaluated in blood of these animals. Some parameters were also evaluated in cerebellum from neonatal rats and in cerebellum neuronal cultures. Prist significantly increased malondialdehyde (MDA) levels and carbonyl formation and reduced sulfhydryl content and glutathione (GSH) concentrations in cerebellum of young rats. It also caused DNA strand damage in cerebellum and induced a high micronuclei frequency in blood. On the other hand, this fatty acid significantly reduced α-ketoglutarate dehydrogenase and aconitase activities in rat cerebellum. We also verified that Prist-induced increase of MDA levels was totally prevented by melatonin and attenuated by α-tocopherol but not by the nitric oxide synthase inhibitor N(ω)-nitro-L-arginine methyl ester, indicating the involvement of reactive oxygen species in this effect. Cerebellum from neonate rats also showed marked alterations of redox homeostasis, including an increase of MDA levels and a decrease of sulfhydryl content and GSH concentrations elicited by Prist. Finally, Prist provoked an increase of dichlorofluorescein (DCFH) oxidation in cerebellum-cultivated neurons. Our present data indicate that Prist compromises redox homeostasis in rat cerebellum and blood and inhibits critical enzymes of the citric acid cycle that are susceptible to free radical attack. The present findings may contribute to clarify the pathogenesis of the cerebellar alterations observed in patients affected by ZS and some peroxisomal disorders in which Prist is accumulated.

Influence of Zika virus on the cytotoxicity, cell adhesion, apoptosis and inflammatory markers of glioblastoma cells.

Glioblastoma (GBM) is one of the most common types of brain tumor in adults. Despite the availability of treatments for this disease, GBM remains one of the most lethal and difficult types of tumors to treat, and thus, a majority of patients die within 2 years of diagnosis. Infection with Zika virus (ZIKV) inhibits cell proliferation and induces apoptosis, particularly in developing neuronal cells, and thus could potentially be considered an alternative for GBM treatment. In the present study, two GBM cell lines (U-138 and U-251) were infected with ZIKV at different multiplicities of infection (0.1, 0.01 and 0.001), and cell viability, migration, adhesion, induction of apoptosis, interleukin levels and CD14/CD73 cell surface marker expression were analyzed. The present study demonstrated that ZIKV infection promoted loss of cell viability and increased apoptosis in U-138 cells, as measured by MTT and triplex assay, respectively. Changes in cell migration, as determined by wound healing assay, were not observed; however, the GBM cell lines exhibited an increase in cell adhesion when compared with non-tumoral cells (Vero). The Luminex immunoassay showed a significant increase in the expression levels of IL-4 specifically in U-251 cells (MOI 0.001) following exposure to ZIKV. There was no significant change in the expression levels of IFN-γ upon ZIKV infection in the cell lines tested. Furthermore, a marked increase in the percentage of cells expressing the CD14 surface marker was observed in both GBM cell lines compared with in Vero cells; and significantly increased CD73 expression was observed particularly in U-251 cells, when compared with uninfected cells. These findings indicate that ZIKV infection could lead to reduced cell viability, elevated CD73 expression, improved cellular adherence, and higher rates of apoptosis in glioblastoma cells. Further studies are required to explore the potential use of ZIKV in the treatment of GBM.

Marked inhibition of Na+, K(+)- ATPase activity and the respiratory chain by phytanic acid in cerebellum from young rats: possible underlying mechanisms of cerebellar ataxia in Refsum disease.

Refsum disease is an autosomal recessive disorder of peroxisomal metabolism biochemically characterized by highly elevated concentrations of phytanic acid (Phyt) in a variety of tissues including the cerebellum. Reduction of plasma Phyt levels by dietary restriction intake ameliorates ataxia, a common clinical manifestation of this disorder, suggesting a neurotoxic role for this branched-chain fatty acid. Therefore, considering that the underlying mechanisms of cerebellum damage in Refsum disease are poorly known, in the present study we tested the effects of Phyt on important parameters of bioenergetics, such as the activities of the respiratory chain complexes I to IV, creatine kinase and Na(+), K(+)- ATPase in cerebellum preparations from young rats. The activities of complexes I, II, I-III and II-III and Na(+), K(+)- ATPase were markedly inhibited (65-85%) in a dose-dependent manner by Phyt. In contrast, creatine kinase and complex IV activities were not altered by this fatty acid. Therefore, it is presumed that impairment of the electron flow through the respiratory chain and inhibition of Na(+), K(+)- ATPase that is crucial for synaptic function may be involved in the pathophysiology of the cerebellar abnormalities manifested as ataxia in Refsum disease and in other peroxisomal disorders in which brain Phyt accumulates.

Neurochemical evidence that the metabolites accumulating in 3-methylcrotonyl-CoA carboxylase deficiency induce oxidative damage in cerebral cortex of young rats.

Isolated 3-methylcrotonyl-CoA carboxylase deficiency (3MCCD) is an autosomal recessive disorder of leucine metabolism biochemically characterized by accumulation of 3-methylcrotonylglycine (3MCG), 3-methylcrotonic acid (3MCA) and 3-hydroxyisovaleric acid. A considerable number of affected individuals present neurological symptoms with or without precedent crises of metabolic decompensation and brain abnormalities whose pathogenesis is poorly known. We investigated the in vitro effects of 3MCG and 3MCA on important parameters of oxidative stress in cerebral cortex of young rats. 3MCG and 3MCA significantly increased TBA-RS and carbonyl formation, indicating that these compounds provoke lipid and protein oxidation, respectively. In contrast, nitric oxide production was not affected by 3MCG and 3MCA. Furthermore, 3MCG- and 3MCA-induced elevation of TBA-RS values was fully prevented by melatonin, trolox and reduced glutathione, but not by the nitric oxide inhibitor N(ω)-nitro-L-arginine methyl ester or the combination of catalase plus superoxide dismutase, indicating that reactive oxygen species were involved in the oxidative damage caused by these compounds. We also found that the activity of the antioxidant enzymes glutathione peroxidase, catalase, superoxide dismutase and glutathione reductase were not altered in vitro by 3MCG and 3MCA. It is therefore presumed that alterations of the cellular redox homeostasis caused by the major metabolites accumulating in 3MCCD may potentially be involved in the pathophysiology of the neurological dysfunction and structural brain alterations found in patients affected by this disorder.

Disruption of mitochondrial homeostasis by phytanic acid in cerebellum of young rats.

Phytanic acid (Phyt) brain concentrations are highly increased in Refsum disease, a peroxisomal disorder clinically characterized by neurological features, cardiac abnormalities, and retinitis pigmentosa. Considering that the pathogenesis of cerebellar ataxia, a common finding in this disease, is still unknown, in the present work we investigated the in vitro effects of Phyt at concentrations similar to those found in affected patients on important parameters of mitochondrial homeostasis in cerebellum from young rats. The respiratory parameters states 3 and 4 and respiratory control ratio (RCR) determined by oxygen consumption, membrane potential (∆Ψm), NAD(P)H pool content, and swelling were evaluated in mitochondrial preparations from this cerebral structure. Phyt markedly increased state 4 respiration, whereas state 3 respiration, the RCR, the mitochondrial matrix NAD(P)H content, and ∆Ψm were decreased by this fatty acid, being the latter effect partially prevented by N-acetylcysteine. These data indicate that Phyt behaves as an uncoupler of oxidative phosphorylation and as a metabolic inhibitor disrupting mitochondrial homeostasis in cerebellum. It is proposed that these pathomechanisms may contribute at least in part to the cerebellar alterations found in Refsum disease.

Reduction of Na+, K+-ATPase activity and expression in cerebral cortex of glutaryl-CoA dehydrogenase deficient mice: a possible mechanism for brain injury in glutaric aciduria type I.

Mitochondrial dysfunction has been proposed to play an important role in the neuropathology of glutaric acidemia type I (GA I). However, the relevance of bioenergetics disruption and the exact mechanisms responsible for the cortical leukodystrophy and the striatum degeneration presented by GA I patients are not yet fully understood. Therefore, in the present work we measured the respiratory chain complexes activities I-IV, mitochondrial respiratory parameters state 3, state 4, the respiratory control ratio and dinitrophenol (DNP)-stimulated respiration (uncoupled state), as well as the activities of α-ketoglutarate dehydrogenase (α-KGDH), creatine kinase (CK) and Na+, K+-ATPase in cerebral cortex, striatum and hippocampus from 30-day-old Gcdh-/- and wild type (WT) mice fed with a normal or a high Lys (4.7%) diet. When a baseline (0.9% Lys) diet was given, we verified mild alterations of the activities of some respiratory chain complexes in cerebral cortex and hippocampus, but not in striatum from Gcdh-/- mice as compared to WT animals. Furthermore, the mitochondrial respiratory parameters and the activities of α-KGDH and CK were not modified in all brain structures from Gcdh-/- mice. In contrast, we found a significant reduction of Na(+), K(+)-ATPase activity associated with a lower degree of its expression in cerebral cortex from Gcdh-/- mice. Furthermore, a high Lys (4.7%) diet did not accentuate the biochemical alterations observed in Gcdh-/- mice fed with a normal diet. Since Na(+), K(+)-ATPase activity is required for cell volume regulation and to maintain the membrane potential necessary for a normal neurotransmission, it is presumed that reduction of this enzyme activity may represent a potential underlying mechanism involved in the brain swelling and cortical abnormalities (cortical atrophy with leukodystrophy) observed in patients affected by GA I.

Marked reduction of Na(+), K(+)-ATPase and creatine kinase activities induced by acute lysine administration in glutaryl-CoA dehydrogenase deficient mice.

Glutaric acidemia type I (GA I) is an inherited neurometabolic disorder caused by a severe deficiency of the mitochondrial glutaryl-CoA dehydrogenase activity leading to accumulation of predominantly glutaric (GA) and 3-hydroxyglutaric (3HGA) acids in the brain and other tissues. Affected patients usually present with hypotonia and brain damage and acute encephalopathic episodes whose pathophysiology is not yet fully established. In this study we investigated important parameters of cellular bioenergetics in brain, heart and skeletal muscle from 15-day-old glutaryl-CoA dehydrogenase deficient mice (Gcdh(-/-)) submitted to a single intra-peritoneal injection of saline (Sal) or lysine (Lys - 8 µmol/g) as compared to wild type (WT) mice. We evaluated the activities of the respiratory chain complexes II, II-III and IV, α-ketoglutarate dehydrogenase (α-KGDH), creatine kinase (CK) and synaptic Na(+), K(+)-ATPase. No differences of all evaluated parameters were detected in the Gcdh(-/-) relatively to the WT mice injected at baseline (Sal). Furthermore, mild increases of the activities of some respiratory chain complexes (II-III and IV) were observed in heart and skeletal muscle of Gcdh(-/-) and WT mice after Lys administration. However, the most marked effects provoked by Lys administration were marked decreases of the activities of Na(+), K(+)-ATPase in brain and CK in brain and skeletal muscle of Gcdh(-/-) mice. In contrast, brain α-KGDH activity was not altered in WT and Gcdh(-/-) injected with Sal or Lys. Our results demonstrate that reduction of Na(+), K(+)-ATPase and CK activities may play an important role in the pathogenesis of the neurodegenerative changes in GA I.

3-Methylcrotonylglycine disrupts mitochondrial energy homeostasis and inhibits synaptic Na(+),K (+)-ATPase activity in brain of young rats.

Deficiency of 3-methylcrotonyl-CoA carboxylase activity is an inherited metabolic disease biochemically characterized by accumulation and high urinary excretion of 3-methylcrotonylglycine (3MCG), and also of 3-hydroisovalerate in lesser amounts. Affected patients usually have neurologic dysfunction, brain abnormalities and cardiomyopathy, whose pathogenesis is still unknown. The present study investigated the in vitro effects of 3MCG on important parameters of energy metabolism, including CO(2) production from labeled acetate, enzyme activities of the citric acid cycle, as well as of the respiratory chain complexes I-IV (oxidative phosphorylation), creatine kinase (intracellular ATP transfer), and synaptic Na(+),K(+)-ATPase (neurotransmission) in brain cortex of young rats. 3MCG significantly reduced CO(2) production, implying that this compound compromises citric acid cycle activity. Furthermore, 3MCG diminished the activities of complex II-III of the respiratory chain, mitochondrial creatine kinase and synaptic membrane Na(+),K(+)-ATPase. Furthermore, antioxidants were able to attenuate or fully prevent the inhibitory effect of 3MCG on creatine kinase and synaptic membrane Na(+),K(+)-ATPase activities. We also observed that lipid peroxidation was elicited by 3MCG, suggesting the involvement of free radicals on 3MCG-induced effects. Considering the importance of the citric acid cycle and the electron flow through the respiratory chain for brain energy production, creatine kinase for intracellular energy transfer, and Na(+),K(+)-ATPase for the maintenance of the cell membrane potential, the present data indicate that 3MCG potentially impairs mitochondrial brain energy homeostasis and neurotransmission. It is presumed that these pathomechanisms may be involved in the neurological damage found in patients affected by 3-methylcrotonyl-CoA carboxylase deficiency.

Phytanic acid disturbs mitochondrial homeostasis in heart of young rats: a possible pathomechanism of cardiomyopathy in Refsum disease.

Phytanic acid (Phyt) accumulates in tissues and biological fluids of patients affected by Refsum disease. Although cardiomyopathy is an important clinical manifestation of this disorder, the mechanisms of heart damage are poorly known. In the present study, we investigated the in vitro effects of Phyt on important parameters of oxidative stress in heart of young rats. Phyt significantly increased thiobarbituric acid-reactive substances levels (P < 0.001) and carbonyl formation (P < 0.01), indicating that this fatty acid induces lipid and protein oxidative damage, respectively. In contrast, Phyt did not alter sulfhydryl oxidation. Phyt also decreased glutathione (GSH) concentrations (P < 0.05), an important non-enzymatic antioxidant defense. Moreover, Phyt increased 2',7'-dichlorofluorescin oxidation (DCFH) (P < 0.01), reflecting increased reactive species generation. We also found that the induced lipid and protein oxidative damage, as well as the decreased GSH levels and increased DCFH oxidation provoked by this fatty acid were prevented or attenuated by the reactive oxygen species scavengers melatonin, trolox, and GSH, but not by the nitric oxide inhibitor N: (ω)-nitro-L: -arginine methyl ester, suggesting that reactive oxygen species were involved in these effects. Next, we verified that Phyt strongly inhibited NADH-cytochrome c oxidoreductase (complex I-III) activity (P < 0.001) in heart supernatants, and decreased membrane potential and the NAD(P)H pool in heart mitochondria, indicating that Phyt acts as a metabolic inhibitor and as an uncoupler of the electron transport chain. Therefore, it can be presumed that disturbance of cellular energy and redox homeostasis induced by Phyt may possibly contribute to the cardiomyopathy found in patients affected by Refsum disease.

Chronic postnatal ornithine administration to rats provokes learning deficit in the open field task.

Hyperornithinemia is the biochemical hallmark of hyperornithinemia-hyperammonemia-homocitrullinuria (HHH) syndrome, an inherited metabolic disease clinically characterized by mental retardation whose pathogenesis is still poorly known. In the present work, we produced a chemical animal model of hyperornithinemia induced by a subcutaneous injection of saline-buffered Orn (2-5 µmol/g body weight) to rats. High brain Orn concentrations were achieved, indicating that Orn is permeable to the blood brain barrier. We then investigated the effect of early chronic postnatal administration of Orn on physical development and on the performance of adult rats in the open field, the Morris water maze and in the step down inhibitory avoidance tasks. Chronic Orn treatment had no effect on the appearance of coat, eye opening or upper incisor eruption, nor on the free-fall righting reflex and on the adult rat performance in the Morris water maze and in the inhibitory avoidance tasks, suggesting that physical development, aversive and spatial localization were not changed by Orn. However, Orn-treated rats did not habituate to the open field apparatus, implying a deficit of learning/memory. Motor activity was the same for Orn- and saline- injected animals. We also verified that Orn subcutaneous injections provoked lipid peroxidation in the brain, as determined by a significant increase of thiobarbituric acid-reactive substances levels. Our results indicate that chronic early postnatal hyperornithinemia may impair the central nervous system, causing minor disabilities which result in specific learning deficiencies.

Impairment of brain redox homeostasis caused by the major metabolites accumulating in hyperornithinemia-hyperammonemia-homocitrullinuria syndrome in vivo.

Ornithine, ammonia and homocitrulline are the major metabolites accumulating in hyperornithinemia-hyperammonemia-homocitrullinuria syndrome, a genetic disorder characterized by neurological regression whose pathogenesis is still not understood. The present work investigated the in vivo effects of intracerebroventricular administration of ornithine and homocitrulline in the presence or absence of hyperammonemia induced by intraperitoneal urease treatment on a large spectrum of oxidative stress parameters in cerebral cortex from young rats in order to better understand the role of these metabolites on brain damage. Ornithine increased thiobarbituric acid-reactive substances (TBA-RS) levels and carbonyl formation and decreased total antioxidant status (TAS) levels. We also observed that the combination of hyperammonemia with ornithine resulted in significant decreases of sulfhydryl levels, reduced glutathione (GSH) concentrations and the activities of catalase (CAT) and glutathione peroxidase (GPx), highlighting a synergistic effect of ornithine and ammonia. Furthermore, homocitrulline caused increases of TBA-RS values and carbonyl formation, as well as decreases of GSH concentrations and GPx activity. Hcit with hyperammonemia (urease treatment) decreased TAS and CAT activity. We also showed that urease treatment per se was able to enhance TBA-RS levels. Finally, nitric oxide production was not altered by Orn and Hcit alone or in combination with hyperammonemia. Our data indicate that the major metabolites accumulating in hyperornithinemia-hyperammonemia-homocitrullinuria syndrome provoke lipid and protein oxidative damage and a reduction of the antioxidant defenses in the brain. Therefore, it is presumed that oxidative stress may represent a relevant pathomechanism involved in the brain damage found in patients affected by this disease.

Neurochemical evidence that lysine inhibits synaptic Na+,K+-ATPase activity and provokes oxidative damage in striatum of young rats in vivo.

Lysine (Lys) accumulation in tissues and biological fluids is the biochemical hallmark of patients affected by familial hyperlysinemia (FH) and other inherited metabolic disorders. In the present study we investigated the effects of acute administration of Lys on relevant parameters of energy metabolism and oxidative stress in striatum of young rats. We verified that Lys in vivo intrastriatal injection did not change the citric acid cycle function and creatine kinase activity, but, in contrast, significantly inhibited synaptic Na(+),K(+)-ATPase activity in striatum prepared 2 and 12 h after injection. Moreover, Lys induced lipid peroxidation and diminished the concentrations of glutathione 2 h after injection. These effects were prevented by the antioxidant scavengers melatonin and the combination of α-tocopherol and ascorbic acid. Lys also inhibited glutathione peroxidase activity 12 h after injection. Therefore it is assumed that inhibition of synaptic Na(+),K(+)-ATPase and oxidative damage caused by brain Lys accumulation may possibly contribute to the neurological manifestations of FH and other neurometabolic conditions with high concentrations of this amino acid.

Neuronal Death, Glial Reactivity, Microglia Activation, Oxidative Stress and Bioenergetics Impairment Caused by Intracerebroventricular Administration of D-2-hydroxyglutaric Acid to Neonatal Rats.

D-2-hydroxyglutaric acid (D-2-HG) accumulates and is the biochemical hallmark of D-2-hydroxyglutaric acidurias (D-2-HGA) types I and II, which comprehend two inherited neurometabolic diseases with severe cerebral abnormalities. Since the pathogenesis of these diseases is poorly established, we tested whether D-2-HG could be neurotoxic to neonatal rats. D-2-HG intracerebroventricular administration caused marked vacuolation in cerebral cortex and striatum. In addition, glial fibrillary acidic protein (GFAP), S-100 calcium binding protein B (S100B) and ionized calcium-binding adapter molecule 1 (Iba-1) staining was increased in both brain structures, suggesting glial reactivity and microglial activation. D-2-HG also provoked a reduction of NeuN-positive cells in cerebral cortex, signaling neuronal death. Considering that disturbances in redox homeostasis and energy metabolism may be involved in neuronal damage and glial reactivity, we assessed whether D-2-HG could induce oxidative stress and bioenergetics impairment. D-2-HG treatment significantly augmented reactive oxygen and nitrogen species generation, provoked lipid peroxidation and protein oxidative damage, diminished glutathione concentrations and augmented superoxide dismutase and catalase activities in cerebral cortex. Increased reactive oxygen species generation, lipoperoxidation and protein oxidation were also found in striatum. Furthermore, the antagonist of NMDA glutamate receptor MK-801 and the antioxidant melatonin were able to prevent most of D-2-HG-induced pro-oxidant effects, implying the participation of these receptors in D-2-HG-elicited oxidative damage. Our results also demonstrated that D-2-HG markedly reduced the respiratory chain complex IV and creatine kinase activities. It is presumed that these deleterious pathomechanisms caused by D-2-HGA may be involved in the brain abnormalities characteristic of early-infantile onset D-2-HGA.

Experimental evidence that phenylalanine provokes oxidative stress in hippocampus and cerebral cortex of developing rats.

High levels of phenylalanine (Phe) are the biochemical hallmark of phenylketonuria (PKU), a neurometabolic disorder clinically characterized by severe mental retardation and other brain abnormalities, including cortical atrophy and microcephaly. Considering that the pathomechanisms leading to brain damage and particularly the marked cognitive impairment in this disease are poorly understood, in the present study we investigated the in vitro effect of Phe, at similar concentrations as to those found in brain of PKU patients, on important parameters of oxidative stress in the hippocampus and cerebral cortex of developing rats. We found that Phe induced in vitro lipid peroxidation (increase of TBA-RS values) and protein oxidative damage (sulfhydryl oxidation) in both cerebral structures. Furthermore, these effects were probably mediated by reactive oxygen species, since the lipid oxidative damage was totally prevented by the free radical scavengers alpha-tocopherol and melatonin, but not by L-NAME, a potent inhibitor of nitric oxide synthase. Accordingly, Phe did not induce nitric oxide synthesis, but significantly decreased the levels of reduced glutathione (GSH), the major brain antioxidant defense, in hippocampus and cerebral cortex supernatants. Phe also reduced the thiol groups of a commercial GSH solution in a cell-free medium. We also found that the major metabolites of Phe catabolism, phenylpyruvate, phenyllactate and phenylacetate also increased TBA-RS levels in cerebral cortex, but to a lesser degree. The data indicate that Phe elicits oxidative stress in the hippocampus, a structure mainly involved with learning/memory, and also in the cerebral cortex, which is severely damaged in PKU patients. It is therefore presumed that this pathomechanism may be involved at least in part in the severe cognitive deficit and in the characteristic cortical atrophy associated with dysmyelination and leukodystrophy observed in this disorder.

Inhibition of creatine kinase activity by lysine in rat cerebral cortex.

Accumulation of lysine (Lys) in tissues and biochemical fluids is the biochemical hallmark of patients affected by familial hyperlysinemia (FH) and also by other inherited neurometabolic disorders. In the present study, we investigated the in vitro effect of Lys on various parameters of energy metabolism in cerebral cortex of 30-day-old Wistar rats. We verified that total (tCK) and cytosolic creatine kinase activities were significantly inhibited by Lys, in contrast to the mitochondrial isoform which was not affected by this amino acid. Furthermore, the inhibitory effect of Lys on tCK activity was totally prevented by reduced glutathione, suggesting a possible role of reactive species oxidizing critical thiol groups of the enzyme. In contrast, Lys did not affect (14)CO(2) production from [U-(14)C] glucose (aerobic glycolytic pathway) and [1-(14)C] acetic acid (citric acid cycle activity) neither the various activities of the electron transfer chain and synaptic Na(+)K(+)-ATPase at concentrations as high as 5.0 mM. Considering the importance of creatine kinase (CK) activity for brain energy metabolism homeostasis and especially ATP transfer and buffering, our results suggest that inhibition of this enzyme by Lys may contribute to the neurological signs presented by symptomatic patients affected by FH and other neurodegenerative disorders in which Lys accumulates.

Evidence that folic acid deficiency is a major determinant of hyperhomocysteinemia in Parkinson's disease.

In the present work we measured blood levels of total homocysteine ((t)Hcy), vitamin B(12) and folic acid in patients with Parkinson s disease (PD) and in age-matched controls and searched for possible associations between these levels with smoking, alcohol consumption, L-DOPA treatment and disease duration in PD patients. We initially observed that plasma (t)Hcy levels were increased by around 30 % in patients affected by PD compared to controls. Linear correlation, multiple regression and comparative analyses revealed that the major determinant of the increased plasma concentrations of (t)Hcy in PD patients was folic acid deficiency, whereas in controls (t)Hcy levels were mainly determined by plasma vitamin B(12) concentrations. We also observed that alcohol consumption, gender and L-DOPA treatment did not significantly alter plasma (t)Hcy, folic acid and vitamin B(12) levels in parkinsonians. Furthermore, disease duration was positively associated with (t)Hcy levels and smoking was linked with a deficit of folic acid in PD patients. Considering the potential synergistic deleterious effects of Hcy increase and folate deficiency on the central nervous system, we postulate that folic acid should be supplemented to patients affected by PD in order to normalize blood Hcy and folate levels, therefore potentially avoiding these risk factors for neurologic deterioration in this disorder.