Zika virus-induced brain malformations in chicken embryos.

BACKGROUND: Zika virus (ZIKV) was confirmed to be related to microcephaly in 2016. However, there is still a need for understanding the embryonic morphological changes induced by ZIKV and when they occur. Here, chicken embryos were chosen as experimental model of ZIKV to evaluate virus-associated morphological alterations that might take place during embryonic development. METHODS: A screening with different viral doses was conducted in embryos at HH Stage 10-12 (E1.5) as well as a follow up of the first 5 days postinfection (dpi) was performed to observe the main morphologic changes post ZIKV infection. RESULTS: ZIKV exposed embryos presented a higher prevalence of mortality and defects such as brain malformation when compared to controls. Moreover, we observed that the phenotypes become more evident at 4dpi, when the viral load quantification reaches a peak. CONCLUSIONS: We found that ZIKV exposed embryos presented a high prevalence of mortality and central nervous system (CNS) abnormalities in a dose-dependent manner. The phenotype was more evident 4 days postinfection, when the viral load quantification reached a peak.

The Brazilian-Portuguese version of the Sleep Hygiene Index (SHI): validity, reliability and association with depressive symptoms and sleep-related outcomes.

OBJECTIVE: To translate the Sleep Hygiene Index (SHI) to Brazilian Portuguese, to describe its psychometric properties and to show its association with sleep quality, daytime sleepiness, risk for sleep apnea and depressive symptoms. METHODS: Thirty subjects participated in the cultural adaptation and the item clarity evaluation. Twenty subjects answered the instrument in three different time-points for test-retest reliability. Eighty adult workers completed the SHI, the Pittsburgh Sleep Quality Index (PSQI), the Epworth Sleepiness Scale (ESS), the Beck Depression Inventory (BDI) and the STOP-BANG (S-B). RESULTS: SHI shows an acceptable internal consistency (Cronbach's α=0.75), as well as a high reproducibility (intraclass correlation=0.972, p<0.01). The three final factors of confirmatory factor analysis extract an average of 48.22% of the total sample variance. Worse sleep hygiene (higher SHI score) correlated with poor sleep quality (r=0.398, p<0.001), excessive daytime sleepiness (r=0.406, p<0.001) and depressive symptoms (r=0.324, p=0.003). No correlations with S-B were found. CONCLUSIONS: SHI presents satisfactory-to-optimal psychometric properties. This instrument is useful for treatment planning and management of sleep hygiene practices. Thus, it represents a reliable way of assessing sleep hygiene quantitatively in both research and clinical settings.

Guanosine protects against Ca2+-induced mitochondrial dysfunction in rats.

Mitochondria play an important role in cell life and in the regulation of cell death. In addition, mitochondrial dysfunction contributes to a wide range of neuropathologies. The nucleoside Guanosine (GUO) is an endogenous molecule, presenting antioxidant properties, possibly due to its direct scavenging ability and/or from its capacity to activate the antioxidant defense system. GUO demonstrate a neuroprotective effect due to the modulation of the glutamatergic system and maintenance of the redox system. Thus, considering the few studies focused on the direct effects of GUO on mitochondrial bioenergetics, we designed a study to evaluate the in vitro effects of GUO on rat mitochondrial function, as well as against Ca2+-induced impairment. Our results indicate that GUO prevented mitochondrial dysfunction induced by Ca2+ misbalance, once GUO was able to reduce mitochondrial swelling in the presence of Ca2+, as well as ROS production and hydrogen peroxide levels, and to increase manganese superoxide dismutase activity, oxidative phosphorylation and tricarboxylic acid cycle activities. Our study indicates for the first time that GUO could direct prevent the mitochondrial damage induced by Ca2+ and that these effects were not related to its scavenging properties. Our data indicates that GUO could be included as a new pharmacological strategy for diseases linked to mitochondrial dysfunction.

Neuroprotective Effects of Guanosine Administration on In Vivo Cortical Focal Ischemia in Female and Male Wistar Rats.

Guanosine (GUO) has neuroprotective effects in experimental models of brain diseases involving glutamatergic excitotoxicity in male animals; however, its effects in female animals are poorly understood. Thus, we investigated the influence of gender and GUO treatment in adult male and female Wistar rats submitted to focal permanent cerebral ischemia in the motor cortex brain. Female rats were subdivided into non-estrogenic and estrogenic phase groups by estrous cycle verification. Immediately after surgeries, the ischemic animals were treated with GUO or a saline solution. Open field and elevated plus maze tasks were conducted with ischemic and naïve animals. Cylinder task, immunohistochemistry and infarct volume analyses were conducted only with ischemic animals. Female GUO groups achieved a full recovery of the forelimb symmetry at 28-35 days after the insult, while male GUO groups only partially recovered at 42 days, in the final evaluation. The ischemic insult affected long-term memory habituation to novelty only in female groups. Anxiety-like behavior, astrocyte morphology and infarct volume were not affected. Regardless the estrous cycle, the ischemic injury affected differently female and male animals. Thus, this study points that GUO is a potential neuroprotective compound in experimental stroke and that more studies, considering the estrous cycle, with both genders are recommended in future investigation concerning brain diseases.

Long-term effect of uncomplicated Plasmodium berghei ANKA malaria on memory and anxiety-like behaviour in C57BL/6 mice.

BACKGROUND: Cerebral malaria, the main complication of Plasmodium falciparum infection in humans, is associated with persistent neurocognitive sequels both in human disease and the murine experimental model. In recent years, cognitive deficits related to uncomplicated (non-cerebral) malaria have also been reported in chronically exposed residents of endemic areas, but not in some murine experimental models of non-cerebral malaria. This study aimed at evaluating the influence of uncomplicated malaria on different behavioural paradigms associated with memory and anxiety-like parameters in a murine model that has the ability to develop cerebral malaria. METHODS: Plasmodium berghei ANKA-infected and non-infected C57BL/6 mice were used. Development of cerebral malaria was prevented by chloroquine treatment starting on the fourth day of infection. The control group (non-infected mice) were treated with PBS. The effect of uncomplicated malaria infection on locomotor habituation, short and long-term memory and anxious-like behaviour was evaluated 64 days after parasite clearance in assays including open field, object recognition, Y-maze and light/dark tasks. RESULTS: Plasmodium berghei ANKA-infected mice showed significant long-lasting disturbances reflected by a long-term memory-related behaviour on open field and object recognition tasks, accompanied by an anxious-like phenotype availed on open field and light-dark tasks. CONCLUSIONS: Long-term neurocognitive sequels may follow an uncomplicated malaria episode in an experimental model prone to develop cerebral malaria, even if the infection is treated before the appearance of clinical signs of cerebral impairment.

α-Ketoadipic Acid and α-Aminoadipic Acid Cause Disturbance of Glutamatergic Neurotransmission and Induction of Oxidative Stress In Vitro in Brain of Adolescent Rats.

Tissue accumulation of α-ketoadipic (KAA) and α-aminoadipic (AAA) acids is the biochemical hallmark of α-ketoadipic aciduria. This inborn error of metabolism is currently considered a biochemical phenotype with uncertain clinical significance. Considering that KAA and AAA are structurally similar to α-ketoglutarate and glutamate, respectively, we investigated the in vitro effects of these compounds on glutamatergic neurotransmission in the brain of adolescent rats. Bioenergetics and redox homeostasis were also investigated because they represent fundamental systems for brain development and functioning. We first observed that AAA significantly decreased glutamate uptake, whereas glutamate dehydrogenase activity was markedly inhibited by KAA in a competitive fashion. In addition, AAA and more markedly KAA induced generation of reactive oxygen and nitrogen species (increase of 2',7'-dichloroflurescein (DCFH) oxidation and nitrite/nitrate levels), lipid peroxidation (increase of malondialdehyde concentrations), and protein oxidation (increase of carbonyl formation and decrease of sulfhydryl content), besides decreasing the antioxidant defenses (reduced glutathione (GSH)) and aconitase activity. Furthermore, KAA-induced lipid peroxidation and GSH decrease were prevented by the antioxidants α-tocopherol, melatonin, and resveratrol, suggesting the involvement of reactive species in these effects. Noteworthy, the classical inhibitor of NMDA glutamate receptors MK-801 was not able to prevent KAA-induced and AAA-induced oxidative stress, determined by DCFH oxidation and GSH levels, making unlikely a secondary induction of oxidative stress through overstimulation of glutamate receptors. In contrast, KAA and AAA did not significantly change brain bioenergetic parameters. We speculate that disturbance of glutamatergic neurotransmission and redox homeostasis by KAA and AAA may play a role in those cases of α-ketoadipic aciduria that display neurological symptoms.

Experimental evidence that overexpression of NR2B glutamate receptor subunit is associated with brain vacuolation in adult glutaryl-CoA dehydrogenase deficient mice: A potential role for glutamatergic-induced excitotoxicity in GA I neuropathology.

Glutaric aciduria type I (GA I) is biochemically characterized by accumulation of glutaric and 3-hydroxyglutaric acids in body fluids and tissues, particularly in the brain. Affected patients show progressive cortical leukoencephalopathy and chronic degeneration of the basal ganglia whose pathogenesis is still unclear. In the present work we investigated parameters of bioenergetics and redox homeostasis in various cerebral structures (cerebral cortex, striatum and hippocampus) and heart of adult wild type (Gcdh(+/+)) and glutaryl-CoA dehydrogenase deficient knockout (Gcdh(-/-)) mice fed a baseline chow. Oxidative stress parameters were also measured after acute lysine overload. Finally, mRNA expression of NMDA subunits and GLT1 transporter was determined in cerebral cortex and striatum of these animals fed a baseline or high lysine (4.7%) chow. No significant alterations of bioenergetics or redox status were observed in these mice. In contrast, mRNA expression of the NR2B glutamate receptor subunit and of the GLT1 glutamate transporter was higher in cerebral cortex of Gcdh(-/-) mice. Furthermore, NR2B expression was markedly elevated in striatum of Gcdh(-/-) animals receiving chronic Lys overload. These data indicate higher susceptibility of Gcdh(-/-) mice to excitotoxic damage, implying that this pathomechanism may contribute to the cortical and striatum alterations observed in GA I patients.

Changes in Brain 14-3-3 Proteins in Response to Insulin Resistance Induced by a High Palatable Diet.

The 14-3-3 protein family takes part in a wide range of cellular processes and is expressed in all eukaryotic organisms. In mammals, seven isoforms (ß, ε, η, γ, τ, ζ, and σ) have been identified. 14-3-3 proteins are suggested to modulate the insulin-signaling cascade in the brain. The aim of this study was to investigate whether insulin resistance state induced by high palatable diet modulates expression of the 14-3-3 proteins in brain. Wistar male rats (n = 8) were divided into two experimental groups: insulin resistant (IR), induced by high palatable diet, and control (CO) group. Biochemical parameters (glucose tolerance test and plasma lipid profile) were evaluated after 130 days. Brain structures (cortex and hippocampus) were dissected for evaluation of messenger RNA (mRNA) and protein levels of different 14-3-3 proteins. Statistical analyses included Student t test and Pearson correlation. Significant decrease was observed in Ywhah and in Ywahq mRNA levels in the cortex of IR group, while no changes were observed in the hippocampus. Significant increase of θ isoform was observed in hippocampus IR group by immunodetection, while no differences were detected in the remaining isoforms. Inverse correlation was observed between blood glucose levels in cortex IR group and both Ywhah and Ywhaq mRNA levels. Protein levels of Creb and phosphatidylinositide 3-kinases (PI3K) showed to be increased in the hippocampus. These alterations may be due to a compensatory effect of impaired insulin signaling. We demonstrated differential expression of 14-3-3 isoforms throughout brain regions of rats with IR. As a whole, our results indicate that brain 14-3-3 levels are influenced by different diets.

Disturbance of the glutamatergic system by glutaric acid in striatum and cerebral cortex of glutaryl-CoA dehydrogenase-deficient knockout mice: possible implications for the neuropathology of glutaric acidemia type I.

The role of excitotoxicity on the neuropathology of glutaric acidemia type I (GA I) is still under debate. Therefore, in the present work, we evaluated glutamate uptake by brain slices and glutamate binding to synaptic membranes, as well as glutamine synthetase activity in cerebral cortex and striatum from glutaryl-CoA dehydrogenase deficient (Gcdh(-/-)) mice along development (7, 15, 30 and 60 days of life) in the hopes of clarifying this matter. We also tested the influence of glutaric acid (GA) added exogenously on these parameters. [(3)H]Glutamate uptake was not significantly altered in cerebral cortex and striatum from Gcdh(-/-) mice, as compared to WT mice. However, GA provoked a significant decrease of [(3)H]glutamate uptake in striatum from both WT and Gcdh(-/-) mice older than 7 days. This inhibitory effect was more pronounced in Gcdh(-/-), as compared to WT mice. The use of a competitive inhibitor of glutamate astrocytic transporters indicated that the decrease of [(3)H]glutamate uptake caused by GA was due to the competition between this organic acid and glutamate for the same astrocytic transporter site. We also found that Na(+)-dependent [(3)H]glutamate binding (binding to transporters) was increased in the striatum from Gcdh(-/-) mice and that GA significantly diminished this binding both in striatum and cerebral cortex from Gcdh(-/-), but not from WT mice. Finally, we observed that glutamine synthetase activity was not changed in brain cortex and striatum from Gcdh(-/-) and WT mice and that GA was not able to alter this activity. It is therefore presumed that a disturbance of the glutamatergic neurotransmission system caused by GA may potentially be involved in the neuropathology of GA I, particularly in the striatum.

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.

Cellular senescence induced by prolonged subculture adversely affects glutamate uptake in C6 lineage.

Several researchers have recently used C6 cells to evaluate functional properties of high-affinity glutamate transporters. However, it has been demonstrated that this lineage suffers several morphological and biochemical alterations according to the number of passages in culture. Currently, there are no reports showing whether functional properties of high-affinity glutamate transporters comply with these sub culturing-dependent modifications. The present study aimed to compare the functional properties of high-affinity glutamate transporters expressed in early (EPC6) and late (LPC6) passage C6 cells through a detailed pharmacological and biochemical characterization. Between 60-180 min of L-[(3)H]glu incubation, LPC6 presented an intracellular [(3)H] 55% lower than EPC6. Both cultures showed a time-dependent increase of intracellular [(3)H] reaching maximal levels at 120 min. Cultures incubated with D-[(3)H]asp showed a time-dependent increase of [(3)H] until 180 min. Moreover, LPC6 have a D-[(3)H]asp-derived intracellular [(3)H] 30-45% lower than EPC6 until 120 min. Only EAAT3 was immunodetected in cultures and its total content was equal between them. PMA-stimulated EAAT3 trafficking to membrane increased 50% of L-[(3)H]glu-derived intracellular [(3)H] in EPC6 and had no effect in LPC6. LPC6 displayed characteristics that resemble senescence, such as high ß-Gal staining, cell enlargement and increase of large and regular nuclei. Our results demonstrated that LPC6 exhibited glutamate uptake impairment, which may have occurred due to its inability to mobilize EAAT3 to cell membrane. This profile might be related to senescent process observed in this culture. Our results suggest that LPC6 cells are an inappropriate glial cellular model to investigate the functional properties of high-affinity glutamate transporters.

Increased glutamate receptor and transporter expression in the cerebral cortex and striatum of gcdh-/- mice: possible implications for the neuropathology of glutaric acidemia type I.

We determined mRNA expression of the ionotropic glutamate receptors NMDA (NR1, NR2A and NR2B subunits), AMPA (GluR2 subunit) and kainate (GluR6 subunit), as well as of the glutamate transporters GLAST and GLT1 in cerebral cortex and striatum of wild type (WT) and glutaryl-CoA dehydrogenase deficient (Gchh-/-) mice aged 7, 30 and 60 days. The protein expression levels of some of these membrane proteins were also measured. Overexpression of NR2A and NR2B in striatum and of GluR2 and GluR6 in cerebral cortex was observed in 7-day-old Gcdh-/-. There was also an increase of mRNA expression of all NMDA subunits in cerebral cortex and of NR2A and NR2B in striatum of 30-day-old Gcdh-/- mice. At 60 days of life, all ionotropic receptors were overexpressed in cerebral cortex and striatum of Gcdh-/- mice. Higher expression of GLAST and GLT1 transporters was also verified in cerebral cortex and striatum of Gcdh-/- mice aged 30 and 60 days, whereas at 7 days of life GLAST was overexpressed only in striatum from this mutant mice. Furthermore, high lysine intake induced mRNA overexpression of NR2A, NR2B and GLAST transcripts in striatum, as well as of GluR2 and GluR6 in both striatum and cerebral cortex of Gcdh-/- mice. Finally, we found that the protein expression of NR2A, NR2B, GLT1 and GLAST were significantly greater in cerebral cortex of Gcdh-/- mice, whereas NR2B and GLT1 was similarly enhanced in striatum, implying that these transcripts were translated into their products. These results provide evidence that glutamate receptor and transporter expression is higher in Gcdh-/- mice and that these alterations may be involved in the pathophysiology of GA I and possibly explain, at least in part, the vulnerability of striatum and cerebral cortex to injury in patients affected by GA I.

A randomized, phase 2 clinical trial of lithium carbonate in Machado-Joseph disease.

BACKGROUND: Because lithium exerts neuroprotective effects in preclinical models of polyglutamine disorders, our objective was to assess the safety and efficacy of lithium carbonate (0.5-0.8 milliequivalents per liter) in patients with Machado-Joseph disease (spinocerebellar ataxia type 3 [MJD/SCA3]). METHODS: For this phase 2, single-center, double-blind, parallel, placebo-controlled trial (ClinicalTrials.gov identifier NCT01096082), 62 patients who had MJD/SCA3 with a disease duration ≤10 years and an independent gait were randomly assigned (1:1) to receive either lithium or placebo. RESULTS: After 24 weeks, 169 adverse events were reported, including 50.3% in the lithium group (P = 1.00; primary safety outcome). Sixty patients (31 in the placebo group and 29 in the lithium group) were analyzed for efficacy (intention-to-treat analysis). Mean progression between groups did not differ according to scores on the Neurological Examination Score for the Assessment of Spinocerebellar Ataxia (NESSCA) after 48 weeks (-0.35; 95% confidence interval, -1.7 to 1.0; primary efficacy outcome). The lithium group exhibited minor progression on the PATA speech-rate (P = 0.002), the nondominant Click Test (P = 0.023), the Spinocerebellar Ataxia Functional Index (P = 0.003), and the Composite Cerebellar Functional Score (P = 0.029). CONCLUSIONS: Lithium was safe and well tolerated, but it had no effect on progression when measured using the NESSCA in patients with MJD/SCA3. This slowdown in secondary outcomes deserves further clarification.

Selenium compounds prevent amyloid ß-peptide neurotoxicity in rat primary hippocampal neurons.

Neuropathological hallmarks of Alzheimer's disease (AD) include amyloid plaque formation, neurofibrillary tangles, neuronal and synaptic loss. This study aims to identify the neuroprotective effects of the selenium compounds on the neurotoxicity of amyloid ß(1-42) in primary cultures of murine hippocampal neurons. Samples were subjected to immunocytochemistry and western blotting techniques to determine the role of treatments on neuronal viability and synaptic protein SNAP-25. We observed a reduced cell viability amyloid ß-peptide (1-42)-induced. When cells were co-treated with amyloid ß-peptide (1-42) and selenium compounds, we verified a strong increase in relative cell viability and in the level of synaptic marker synaptosomal-associated protein SNAP-25 induced by selenium compounds.

Disruption of brain redox homeostasis in glutaryl-CoA dehydrogenase deficient mice treated with high dietary lysine supplementation.

Deficiency of glutaryl-CoA dehydrogenase (GCDH) activity or glutaric aciduria type I (GA I) is an inherited neurometabolic disorder biochemically characterized by predominant accumulation of glutaric acid and 3-hydroxyglutaric acid in the brain and other tissues. Affected patients usually present acute striatum necrosis during encephalopathic crises triggered by metabolic stress situations, as well as chronic leukodystrophy and delayed myelination. Considering that the mechanisms underlying the brain injury in this disease are not yet fully established, in the present study we investigated important parameters of oxidative stress in the brain (cerebral cortex, striatum and hippocampus), liver and heart of 30-day-old GCDH deficient knockout (Gcdh(-/-)) and wild type (WT) mice submitted to a normal lysine (Lys) (0.9% Lys), or high Lys diets (2.8% or 4.7% Lys) for 60 h. It was observed that the dietary supplementation of 2.8% and 4.7% Lys elicited noticeable oxidative stress, as verified by an increase of malondialdehyde concentrations (lipid oxidative damage) and 2-7-dihydrodichlorofluorescein (DCFH) oxidation (free radical production), as well as a decrease of reduced glutathione levels and alteration of various antioxidant enzyme activities (antioxidant defenses) in the cerebral cortex and the striatum, but not in the hippocampus, the liver and the heart of Gcdh(-/-) mice, as compared to WT mice receiving the same diets. Furthermore, alterations of oxidative stress parameters in the cerebral cortex and striatum were more accentuated in symptomatic, as compared to asymptomatic Gcdh(-/-) mice exposed to 4.7% Lys overload. Histopathological studies performed in the cerebral cortex and striatum of these animals exposed to high dietary Lys revealed increased expression of oxidative stress markers despite the absence of significant structural damage. The results indicate that a disruption of redox homeostasis in the cerebral cortex and striatum of young Gcdh(-/-) mice exposed to increased Lys diet may possibly represent an important pathomechanism of brain injury in GA I patients under metabolic stress.

Neurodevelopmental and cognitive behavior of glutaryl-CoA dehydrogenase deficient knockout mice.

AIMS: The establishment of a genetic knockout murine model of glutaric acidemia type I (GAI) with complete loss of glutaryl-CoA dehydrogenase (GCDH) activity has been used to investigate the pathological mechanisms underlying neurological symptoms in this disorder. However, very little has been reported on the neurobehavior of GCDH deficient mice (Gcdh(-/-)). MAIN METHODS: In the present study we evaluated physical (body and weight gain) and neuromotor development (appearance of coat, upper incisor eruption, eye-opening day, motor coordination, muscular strength and climbing), as well as cognitive behavior (inhibitory avoidance) in Gcdh(-/-), as compared to wild type (WT) mice. KEY FINDINGS: We found that Gcdh(-/-) mice did not differ in body and weight gain, appearance of coat, upper incisor eruption, motor coordination and muscular strength, but had a significant delayed eye opening, implying a mild impairment of neurodevelopment in these animals. Furthermore, the climbing behavior was significantly higher in Gcdh(-/-) as compared to WT mice, suggesting an altered dopaminergic function. Finally, Gcdh(-/-) mice presented a deficit of short- and long-term memories in the inhibitory avoidance task. SIGNIFICANCE: Although it is difficult to extrapolate the present findings to the human condition, our present data are particularly interesting in view of the psychomotor/mental delay that occurs in a significant number of GAI patients with no previous history of acute encephalopathy with striatum destruction. Strict and early treatment possibly associated with novel therapies seems therefore important to prevent learning/memory disabilities in GAI patients.

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.

Induction of oxidative stress in brain of glutaryl-CoA dehydrogenase deficient mice by acute lysine administration.

In the present work we evaluated a variety of indicators of oxidative stress in distinct brain regions (striatum, cerebral cortex and hippocampus), the liver, and heart of 30-day-old glutaryl-CoA dehydrogenase deficient (Gcdh(-/-)) mice. The parameters evaluated included thiobarbituric acid-reactive substances (TBA-RS), 2-7-dihydrodichlorofluorescein (DCFH) oxidation, sulfhydryl content, and reduced glutathione (GSH) concentrations. We also measured the activities of the antioxidant enzymes glutathione peroxidase (GPx), glutathione reductase (GR), catalase (CAT), superoxide dismutase (SOD) and glucose-6-phosphate dehydrogenase (G6PD). Under basal conditions glutaric (GA) and 3-OH-glutaric (3OHGA) acids were elevated in all tissues of the Gcdh(-/-) mice, but were essentially absent in WT animals. In contrast there were no differences between WT and Gcdh(-/-) mice in any of the indicators or oxidative stress under basal conditions. Following a single intra-peritoneal (IP) injection of lysine (Lys) there was a moderate increase of brain GA concentration in Gcdh(-/-) mice, but no change in WT. Lys injection had no effect on brain 3OHGA in either WT or Gcdh(-/-) mice. The levels of GA and 3OHGA were approximately 40% higher in striatum compared to cerebral cortex in Lys-treated mice. In the striatum, Lys administration provoked a marked increase of lipid peroxidation, DCFH oxidation, SOD and GR activities, as well as significant reductions of GSH levels and GPx activity, with no alteration of sulfhydryl content, CAT and G6PD activities. There was also evidence of increased lipid peroxidation and SOD activity in the cerebral cortex, along with a decrease of GSH levels, but to a lesser extent than in the striatum. In the hippocampus only mild increases of SOD activity and DCFH oxidation were observed. In contrast, Lys injection had no effect on any of the parameters of oxidative stress in the liver or heart of Gcdh(-/-) or WT animals. These results indicate that in Gcdh(-/-) mice cerebral tissue, particularly the striatum, is at greater risk for oxidative stress than peripheral tissues following Lys administration.

High-glucose and S100B stimulate glutamate uptake in C6 glioma cells.

Diabetes mellitus is a disease associated with several changes in the central nervous system, including oxidative stress and abnormal glutamatergic neurotransmission, and the astrocytes play an essential role in these alterations. In vitro studies of astroglial function have been performed using cultures of primary astrocytes or C6 glioma cells. Herein, we investigated glutamate uptake, glutamine synthetase and S100B secretion in C6 glioma cells cultured in a high-glucose environment, as well as some parameters of oxidative stress and damage. C6 glioma cells, cultured in 12 mM glucose medium, exhibited signals of oxidative and nitrosative stress similar to those found in diabetes mellitus and other models of diabetic disease (decrease in glutathione, elevated NO, DNA damage). Interestingly, we found an increase in glutamate uptake and S100B secretion, and a decrease in glutamine synthetase, which might be linked to the altered glutamatergic communication in diabetes mellitus. Moreover, glutamate uptake in C6 glioma cells, like primary astrocytes, was stimulated by extracellular S100B. Aminoguanidine partially prevented the glial alterations induced by the 12 mM glucose medium. Together, these data emphasize the relevance of astroglia in diabetes mellitus, as well as the importance of glial parameters in the evaluation of diabetic disease progression and treatment.