Tania Marcourakis

Peripheral Oxidative Stress Biomarkers in Mild Cognitive Impairment and Alzheimer's Disease

Oxidative stress has been associated with normal aging and Alzheimers disease (AD). However, little is known about oxidative stress in mild cognitive impairment (MCI) patients who present a high risk for developing AD. The aim of this study was to investigate plasma production of the lipid peroxidation marker, malonaldehyde (MDA) and to determine, in erythrocytes, the enzymatic antioxidant activity of catalase, glutathione peroxidase (GPx), glutathione reductase (GR), and glutathione S-transferase (GST) in 33 individuals with MCI, 29 with mild probable AD and 26 healthy aged subjects. GR/GPx activity ratio was calculated to better assess antioxidant defenses. The relationship between oxidative stress and cognitive performance was also evaluated by the Mini Mental State Examination (MMSE). AD patients showed higher MDA levels than both MCI and healthy elderly subjects. MCI subjects also exhibited higher MDA levels compared to controls. Catalase and GPx activity were similar in MCI and healthy individuals but higher in AD. GR activity was lower in MCI and AD patients than in healthy aged subjects. Additionally, GR/GPx ratio was higher in healthy aged subjects, intermediate in MCI and lower in AD patients. No differences in GST activity were detected among the groups. MMSE was negatively associated with MDA levels (r = -0.31, p = 0.028) and positively correlated with GR/GPx ratio in AD patients (r = 0.68, p < 0.001). MDA levels were also negatively correlated to GR/GPx ratio (r = -0.31, p = 0.029) in the AD group. These results suggest that high lipid peroxidation and decreased antioxidant defenses may be present early in cognitive disorders.

Oxidative state in platelets and erythrocytes in aging and Alzheimer's disease

Several studies have shown involvement of peroxynitrite anion, a potent oxidative agent, in Alzheimers disease (AD) neuropathology. Herein, we assessed in platelets and erythrocytes of AD patients, age-matched and young adults controls: thiobarbituric acid-reactive substances (TBARS) production; superoxide dismutase (SOD), nitric oxide synthase (NOS) and Na,K-ATPase activities; cyclic GMP (cGMP) content, both basal and after sodium nitroprusside (SNP) stimulation. Aging was associated with an increase in TBARS production and NOS activity, a decrease in basal cGMP content and no change in SOD and Na,K-ATPase activities. AD patients, compared to aged controls, have: increase in TBARS production and in NOS, SOD and Na,K-ATPase activities but no alteration in basal cGMP content. SNP increased cGMP platelets production in all groups. In conclusion, we demonstrated in platelets and erythrocytes a disruption in systemic modulation of oxidative stress in aging and with more intensity in AD.

MK-801 and 7-Ni attenuate the activation of brain NF-κB induced by LPS

The activation of nuclear factor-kappaB (NF-kappaB) leads to an increase in the expression of genes involved in important events in the central nervous system (CNS), such as development, plasticity and inflammation. It has been shown that inflammatory stimulus in the brain increases excitatory glutamatergic transmission, especially at N-methyl-D-aspartate (NMDA) receptor. These receptors have an important role in glutamate neurotoxicity and are in general coupled with the generation of nitric oxide (NO) through the activation of neuronal nitric oxide synthase (NOS). We have investigated the involvement of NMDA-NO pathway in LPS induction of NF-kappaB in CNS. Our results demonstrate that systemic LPS activates NF-kappaB in several regions of the CNS, which was partially reduced by the NMDA receptor antagonist dizolcipine (MK-801) and by the selective brain NOS inhibitor 7-Nitroindazol (7-Ni). 7-Ni effects were not synergic to MK-801 effects, suggesting that these compounds act through the same pathway. Dexamethasone caused a stronger reduction in LPS induction of NF-kappaB in CNS, demonstrating that MK-801 and 7-Ni act on a pathway that is responsible only by a fraction of the overall NF-kappaB activation. These results suggest that a considerable part of NF-kappaB activation by LPS is linked to the NMDA/NO pathway in CNS.

Protective effects of aerobic exercise on acute lung injury induced by LPS in mice

IntroductionThe regular practice of physical exercise has been associated with beneficial effects on various pulmonary conditions. We investigated the mechanisms involved in the protective effect of exercise in a model of lipopolysaccharide (LPS)-induced acute lung injury (ALI).MethodsMice were divided into four groups: Control (CTR), Exercise (Exe), LPS, and Exercise + LPS (Exe + LPS). Exercised mice were trained using low intensity daily exercise for five weeks. LPS and Exe + LPS mice received 200 µg of LPS intratracheally 48 hours after the last physical test. We measured exhaled nitric oxide (eNO); respiratory mechanics; neutrophil density in lung tissue; protein leakage; bronchoalveolar lavage fluid (BALF) cell counts; cytokine levels in BALF, plasma and lung tissue; antioxidant activity in lung tissue; and tissue expression of glucocorticoid receptors (Gre).ResultsLPS instillation resulted in increased eNO, neutrophils in BALF and tissue, pulmonary resistance and elastance, protein leakage, TNF-alpha in lung tissue, plasma levels of IL-6 and IL-10, and IL-1beta, IL-6 and KC levels in BALF compared to CTR (P ≤0.02). Aerobic exercise resulted in decreases in eNO levels, neutrophil density and TNF-alpha expression in lung tissue, pulmonary resistance and elastance, and increased the levels of IL-6, IL-10, superoxide dismutase (SOD-2) and Gre in lung tissue and IL-1beta in BALF compared to the LPS group (P ≤0.04).ConclusionsAerobic exercise plays important roles in protecting the lungs from the inflammatory effects of LPS-induced ALI. The effects of exercise are mainly mediated by the expression of anti-inflammatory cytokines and antioxidants, suggesting that exercise can modulate the inflammatory-anti-inflammatory and the oxidative-antioxidative balance in the early phase of ALI.

Age-related changes in cyclic GMP and PKG-stimulated cerebellar Na,K-ATPase activity

Energy deficiency and dysfunction of the Na,K-ATPase are common consequences of many pathological insults. Glutamate through cyclic GMP and cyclic GMP-dependent protein kinase (PKG) has been shown to stimulate alpha(2/3)-Na,K-ATPase activity in the central nervous system. Thus, a slight impairment of this pathway may amplify the disruption of ion homeostasis in the presence of a non-lethal insult. We investigate the effect of aging (4, 12 and 24 months) on the glutamate-cyclic GMP-PKG modulation of alpha1, alpha(2/3)-Na,K-ATPase activity in rat cerebellum and the stimulation of the glutamate-cyclic GMP-PKG pathway at different levels. Cyclic GMP levels and alpha(2/3)-Na,K-ATPase activity were progressively decreased from 4 and 24 month-old animals. However, PKG basal activity was reduced between 4 and 12 months, and no additional change was observed at 24 months. The ability of 8-Br-cyclic GMP to stimulate PKG activity was only reduced between 12 and 24 months. Moreover, glutamate or 8-Br-cyclic GMP promoted a smaller increase of alpha(2/3)-Na,K-ATPase activity at 24 months, when compared to 4 and 12 months. In spite of the age-related reduced basal levels of cyclic GMP, the production induced by CO or NO was not age-related. Finally, inhibition of PKG activation by KT5823 revealed a lower sensitivity of the enzyme at the older age. Taken together, these data show that basal age-related decline in sodium pump activity is a consequence of changes in different steps of the cyclic GMP-PKG pathway. On the other hand, age-related reduction in glutamate positive modulation of cerebellar alpha(2/3)-Na,K-ATPase is linked to a defective PKG signaling pathway.

Neurotoxicity of Anhydroecgonine Methyl Ester, a Crack Cocaine Pyrolysis Product

Smoking crack cocaine involves the inhalation of cocaine and its pyrolysis product, anhydroecgonine methyl ester (AEME). Although there is evidence that cocaine is neurotoxic, the neurotoxicity of AEME has never been evaluated. AEME seems to have cholinergic agonist properties in the cardiovascular system; however, there are no reports on its effects in the central nervous system. The aim of this study was to investigate the neurotoxicity of AEME and its possible cholinergic effects in rat primary hippocampal cell cultures that were exposed to different concentrations of AEME, cocaine, and a cocaine-AEME combination. We also evaluated the involvement of muscarinic cholinergic receptors in the neuronal death induced by these treatments using concomitant incubation of the cells with atropine. Neuronal injury was assessed using 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) and lactate dehydrogenase (LDH) assays. The results of the viability assays showed that AEME is a neurotoxic agent that has greater neurotoxic potential than cocaine after 24 and 48 h of exposure. We also showed that incubation for 48 h with a combination of both compounds in equipotent concentrations had an additive neurotoxic effect. Although both substances decreased cell viability in the MTT assay, only cocaine increased LDH release. Caspase-3 activity was increased after 3 and 6 h of incubation with 1mM cocaine and after 6 h of 0.1 and 1.0mM AEME exposure. Atropine prevented the AEME-induced neurotoxicity, which suggests that muscarinic cholinergic receptors are involved in AEMEs effects. In addition, binding experiments confirmed that AEME has an affinity for muscarinic cholinergic receptors. Nevertheless, atropine was not able to prevent the neurotoxicity produced by cocaine and the cocaine-AEME combination, suggesting that these treatments activated other neuronal death pathways. Our results suggest a higher risk for neurotoxicity after smoking crack cocaine than after cocaine use alone.

Nitric oxide modulates Na+, K+-ATPase activity through cyclic GMP pathway in proximal rat trachea

The present work demonstrated that nitric oxide (NO) modulates Na+, K+-ATPase activity in the proximal rat trachea. Sodium nitroprusside induced concentration-dependent (10-100 microM) stimulation in proximal trachea Na+, K+-ATPase activity. The effect was specific for Na+, K+-ATPase since Mg-ATPase activity was unaffected. This NO-donor changed neither Na+, K+-ATPase nor Mg-ATPase activity in the distal segment. The modulatory action on Na+, K+-ATPase induced by sodium nitroprusside was linked to an increase in nitrates/nitrites and cyclic GMP levels in proximal segments. Modulation of proximal Na+, K+-ATPase activity by sodium nitroprusside was mimicked by S-nitroso-N-acetylpenicillamine (100 microM) and 8-bromo-cyclic GMP (100 microM). Both sodium nitroprusside and 8-bromo-cyclic GMP effects on Na+, K+-ATPase activity of proximal segments of trachea were blocked by 2 microM of KT 5823 (a cyclic GMP-dependent protein kinase inhibitor), but not by 0.5 microM of KT 5720 (a cyclic AMP-dependent protein kinase inhibitor). Both kinase inhibitors decreased proximal Na+, K+-ATPase activity, but did not change Mg-ATPase activity. Okadaic acid (1 microM), a phosphatase-1 inhibitor, increased proximal Na+, K+-ATPase but not Mg-ATPase activity. The effect of okadaic acid was non-additive with that of 8-bromo-cGMP on Na+, K+-ATPase activity. Our results suggest that NO modulates proximal rat trachea Na+, K+-ATPase activity through cyclic GMP and cyclic GMP-dependent protein kinase.

Age-related changes in nitric oxide activity, cyclic GMP, and TBARS levels in platelets and erythrocytes reflect the oxidative status in central nervous system

Aging is associated with an increased susceptibility to neurodegenerative disorders which has been linked to chronic inflammation. This process generates oxygen-reactive species, ultimately responsible for a process known as oxidative stress, leading to changes in nitric oxide (NO), and cyclic guanosine monophosphate (cyclic GMP) signaling pathway. In previous studies, we showed that human aging was associated with an increase in NO Synthase (NOS) activity, a decrease in basal cyclic GMP levels in human platelets, and an increase in thiobarbituric acid-reactant substances (TBARS) in erythrocytes. The aim of the present work was to evaluate NOS activity, TBARS and cyclic GMP levels in hippocampus and frontal cortex and its correlation to platelets and erythrocytes of 4-, 12-, and 24-month-old rats. The result showed an age-related decrease in cyclic GMP levels which was linked to an increase in NOS activity and TBARS in both central areas as well as in platelets and erythrocytes of rats. The present data confirmed our previous studies performed in human platelets and erythrocytes and validate NOS activity and cyclic GMP in human platelet as well as TBARS in erythrocytes as biomarkers to study age-related disorders and new anti-aging therapies.

Vigabatrin: Clinical Evidence Supporting Rational Polytherapy in Management of Uncontrolled Seizures

Summary: Monotherapy is the policy for management of patients with epilepsy. With increasing knowledge of the biology of epilepsy and of the modes of action of antiepileptic drugs (AEDs), this concept must be reevaluated. When monotherapy fails to control seizures, subsequent treatment should be based on “rational pharmacology,” taking into consideration the mode of action of the drugs, to provide improved efficacy with maintained tolerance and ease of administration. Introduction of vigabatrin (VGB) as a new AED calls for just such a reevaluation. VGB is an enzyme‐activated irreversible inhibitor of γ‐aminobutyric acid (GABA)‐transaminase that increases brain and cerebrospinal (CSF) GABA concentrations in animals and humans. It has limited efficacy in the classic animal seizure screening tests, but in many clinical studies has halved the incidence of seizures in ∼50%of patients, especially those with partial epilepsies. We evaluated the efficacy of VGB in “socially integrated and active outpatients” as a likely subset to demonstrate any advantage of rational polytherapy. The criteria for this evaluation included the effects on seizure frequency, patient tolerability, and cognitive performance in a battery of psychometric tests. Fourteen of the 19 patients (73%) completing the study had >50% réduction in seizure frequency, and 10 of 19 (52%)had >70% réduction in seizure frequency. Tolerability appeared good; somnolence was the most frequent adverse event. Three patients complained of a worsening of their seizures, 1 with an increase in frequency and 2 with development of myoclonic jerks not previously reported. No deleterious effect of VGB on cognitive function was noted, and performance in late recognition tests showed significant improvement by the end of the maintenance period, which may have been due to the réduction in seizure frequency. The efficacy of VGB in this study in controlling seizures as compared with results of earlier published studies of refractory epilepsy may be related to selection of patients with more benign partial epilepsy, but does support the need for further studies in such patients to evaluate the hypothesis of rational polytherapy.

O fator de transcrição NF-kapaB nos mecanismos moleculares de ação de psicofármacos

During the last years many efforts have been made in order to elucidate the mechanisms involved in the gene transcription regulation. Special attention has been given to some molecules involved in these regulatory processes, as the transcription factors. Understanding the role of these factors in several neural functions will allow a better knowledge of the disorders related to the central nervous system (CNS) disorders, and will also help identify new pathways for therapeutic access. The transcription factor NF-kB is remarkable for its wide range of actions and also due to the many different proteins involved in its activation. There is evidence this factor works in the plasticity, development and neurodegeneration, and takes part of essential and specific functions of neurons and glial cells. Brain-specific activators of NF-kB include glutamate (via both NMDA and AMPA/KA receptors) and neurotrophins. This article reviews the complex biochemical regulation of NF-B activation, emphasizing the potential of the contributions this transcription factor could make to psychopharmacology field. Therefore, changes in NF-kB activity might provide insight in the development of new psychoactive drugs.