Jeferson Luis Franco

Connecting TNF-α Signaling Pathways to iNOS Expression in a Mouse Model of Alzheimer's Disease: Relevance for the Behavioral and Synaptic Deficits Induced by Amyloid β Protein

Increased brain deposition of amyloid β protein (Aβ) and cognitive deficits are classical signals of Alzheimers disease (AD) that have been highly associated with inflammatory alterations. The present work was designed to determine the correlation between tumor necrosis factor-α (TNF-α)-related signaling pathways and inducible nitric oxide synthase (iNOS) expression in a mouse model of AD, by means of both in vivo and in vitro approaches. The intracerebroventricular injection of Aβ1–40 in mice resulted in marked deficits of learning and memory, according to assessment in the water maze paradigm. This cognition impairment seems to be related to synapse dysfunction and glial cell activation. The pharmacological blockage of either TNF-α or iNOS reduced the cognitive deficit evoked by Aβ1–40 in mice. Similar results were obtained in TNF-α receptor 1 and iNOS knock-out mice. Aβ1–40 administration induced an increase in TNF-α expression and oxidative alterations in prefrontal cortex and hippocampus. Likewise, Aβ1–40 led to activation of both JNK (c-Jun-NH2-terminal kinase)/c-Jun and nuclear factor-κB, resulting in iNOS upregulation in both brain structures. The anti-TNF-α antibody reduced all of the molecular and biochemical alterations promoted by Aβ1–40. These results provide new insights in mouse models of AD, revealing TNF-α and iNOS as central mediators of Aβ action. These pathways might be targeted for AD drug development.

Methylmercury neurotoxicity is associated with inhibition of the antioxidant enzyme glutathione peroxidase

In this study, we investigated the involvement of glutathione peroxidase-GPx in methylmercury (MeHg)-induced toxicity using three models: (a) in mouse brain after treatment with MeHg (40 mg/L in drinking water), (b) in mouse brain mitochondrial-enriched fractions isolated from MeHg-treated animals, and (c) in cultured human neuroblastoma SH-SY5Y cells. First, adult male Swiss mice exposed to MeHg for 21 days showed a significant decrease in GPx activity in the brain and an increase in poly(ADP-ribose) polymerase cleavage, an index of apoptosis. Second, in mitochondrial-enriched fractions isolated from MeHg-treated mice, there was a significant reduction in GPx activity and a concomitant decrease in mitochondrial activity and increases in ROS formation and lipid peroxidation. Incubation of mitochondrial-enriched fractions with mercaptosuccinic acid, a GPx inhibitor, significantly augmented the toxic effects of MeHg administered in vivo. Incubation of mitochondrial-enriched fractions with exogenous GPx completely blocked MeHg-induced mitochondrial lipid peroxidation. Third, SH-SY5Y cells treated for 24 h with MeHg showed a significant reduction in GPx activity. There was a concomitant significant decrease in cell viability and increase in apoptosis. Inhibition of GPx substantially enhanced MeHg toxicity in the SH-SY5Y cells. These results suggest that GPx is an important target for MeHg-induced neurotoxicity, presumably because this enzyme is essential for counteracting the pro-oxidative effects of MeHg both in vitro and in vivo.

Protective effects of Polygala paniculata extract against methylmercury-induced neurotoxicity in mice.

We have examined the possible protective effects of Polygala paniculata extract against methylmercury (MeHg)‐induced neurotoxicity in adult mice. MeHg was diluted in drinking water (40 mg L−1, freely available) and the hydroalcoholic Polygala extract was diluted in a 150 mm NaCl solution and administered by gavage (100 mg kg−1 b.w., twice a day). After a two‐week treatment, MeHg exposure significantly inhibited glutathione peroxidase and increased glutathione reductase activity, while the levels of thiobarbituric acid reactive substances were increased in the cerebral cortex and cerebellum. These alterations were prevented by administration of Polygala extract, except for glutathione reductase activity, which remained elevated in the cerebral cortex. Behavioural interference in the MeHg‐exposed animals was evident through a marked deficit in the motor performance in the rotarod task, which was completely recovered to control levels by Polygala extract co‐administration. This study has shown, for the first time, the in‐vivo protective effects of Polygala extract against MeHg‐induced neurotoxicity. In addition, our findings encourage studies concerning the beneficial effects of P. paniculata on neurological conditions related to excitotoxicity and oxidative stress.

Differential susceptibility following β-amyloid peptide-(1–40) administration in C57BL/6 and Swiss albino mice: Evidence for a dissociation between cognitive deficits and the glutathione system response

Considerable evidence supports the role of oxidative stress in the pathogenesis of Alzheimers disease (AD). Previous studies suggest that the central nervous system (CNS) administration of beta-amyloid peptide, the major constituent of senile plaque in AD, induces oxidative stress in rodents which may contribute to the learning and memory deficits verified in the beta-amyloid model of AD. In the present study, we compared the effects of a single intracerebroventricular (i.c.v.) injection of aggregated beta-amyloid peptide-(1-40) (Abeta(1-40)) (400pmol/mouse) on spatial learning and memory performance, synaptic density and the glutathione (GSH)-dependent antioxidant status in adult male C57BL/6 and Swiss albino mice. Seven days after Abeta(1-40) administration, C57BL/6 and Swiss mice presented similar spatial learning and memory impairments, as evaluated in the water maze task, although these impairments were not found in Abeta(40-1)-treated mice. Moreover, a similar decline of synaptophysin levels was observed in the hippocampus (HC) and prefrontal cortex (PFC) of both Swiss and C57BL/6 mice treated with Abeta(1-40), which suggests synaptic loss. C57BL/6 mice presented lower levels of glutathione-related antioxidant defences (total glutathione (GSH-t) levels, glutathione peroxidase (GPx) and glutathione reductase (GR) activity) in the HC and PFC in comparison to Swiss mice. Despite the reduced basal GSH-dependent antioxidant defences observed in C57BL/6 mice, Abeta(1-40) administration induced significant alterations in the brain antioxidant parameters only in Swiss mice, decreasing GSH-t levels and increasing GPx and GR activity in the HC and PFC 24h after treatment. These results indicate strain differences in the susceptibility to Abeta(1-40)-induced changes in the GSH-dependent antioxidant defences in mice, which should be taken into account in further studies using the Abeta model of AD in mice. In addition, the present findings suggest that the spatial learning and memory deficits induced by beta-amyloid peptides in rodents may not be entirely related to glutathione-dependent antioxidant response.

Involvement of glutathione, ERK1/2 phosphorylation and BDNF expression in the antidepressant-like effect of zinc in rats

We investigated the antidepressant-like effect of zinc chloride (zinc) administered acutely during 7 days (i.p. route), or chronically during 30 days (oral route) in the forced swimming test (FST) in rats. It was also investigated whether the antidepressant-like effect of zinc is associated with changes in the glutathione antioxidant system in the Wistar rat brain. Animals receiving a single zinc dose (5, 15 and 30 mg/kg, i.p.) 24 h prior to analysis showed no changes in the FST, but glutathione reductase and glutathione S-transferase activity were reduced in the hippocampus and cerebral cortex. This treatment did not, however, affect the glutathione status (GSH and GSSG) in both brain structures. The 7-day zinc treatment (1, 5 and 15 mg/kg, i.p.) caused a mild though significant antidepressant-like effect in the FST at the highest dosing, without affecting the glutathione antioxidant system. Finally, a consistent antidepressant-like effect was achieved in the FST after chronic (30 days) zinc treatment (300 mg/L, p.o.). This was accompanied by a significant increase in total glutathione levels in the hippocampus and cerebral cortex. The good response to oral treatment in the FST led us to investigate other variables, such as ERK phosphorylation and BDNF expression. Similar to therapeutic antidepressants, zinc in chronic oral treatment produced an increase in ERK phosphorylation and BDNF expression in the cerebral cortex. It is our hypothesis that up-regulation of neuroprotective effectors (GSH, ERK and BDNF) may be related to the antidepressant properties of zinc, but this will require additional work to be confirmed.

Diphenyl diselenide confers neuroprotection against hydrogen peroxide toxicity in hippocampal slices.

The present study aimed at investigating the potential in vitro protective effect of the organochalcogenide diphenyl diselenide - (PhSe)2 - against hydrogen peroxide (H2O2)-induced toxicity in rat hippocampal slices. Hippocampal slices were treated for 1 h with H2O2 (2 mM) in the presence or absence of (PhSe)2 (0.1-10 microM). H2O2 treatment significantly decreased cell viability (measured by MTT test) and the co-incubation with (PhSe)(2) (10 microM) significantly blunted such phenomenon. The non permeable thiol compounds dithiothreitol (DTT) (100 microM) or reduced glutathione (GSH) (100 microM), which did not display protective effects against H2O2-induced loss of cell viability per se, significantly improved the protective effects elicited by (PhSe)2. Conversely, the permeable form of GSH (GSH monoethyl ester) was unable to alter the neuroprotection mediated by (PhSe)2. The treatment of rat hippocampal slices with H2O2 also increased the lipid peroxidation and decreased the intracellular GSH levels. Moreover, (PhSe)2 (from 0.1 microM) significantly decreased H2O2-induced lipid peroxidation. Interestingly, H2O2 decreased GSH levels and this phenomenon was partially prevented by (PhSe)2. The potential effects of H2O2 on MAPKs phosphorylation (ERK1/2, p38 MAPK and JNK1/2) were also evaluated. Even though H2O2 (2 mM) did not alter p38 MAPK and JNK1/2 phosphorylation in hippocampal slices, it stimulated ERK1/2 phosphorylation and the co-incubation with (PhSe)2 (10 microM) blocked this effect. Taken together, the present results indicate that (PhSe)2 exerts protective effects against H2O2-induced oxidative damage in hippocampal slices and avoided the increase in ERK1/2 phosphorylation promoted by H2O2. The neuroprotective effect of compound seems to be related to its thiol-peroxidase-like activity and appears to occur at the extracellular milieu because a permeable form of GSH was unable to improve the protective effect of the compound as did the impermeable GSH.

Antioxidant and Acetylcholinesterase Response to Repeated Malathion Exposure in Rat Cerebral Cortex and Hippocampus

The wide use of the organophosphate insecticide malathion is accompanied by the risk of human exposure, especially in developing countries, which underlines the need of basic studies in this area. Some reports have shown that low doses of malathion, in a repeated treatment regimen, are unable to reduce acetylcholinesterase (AChE) activity in the rat brain, in contrast to the inhibitory effect in acute treatment. In order to investigate if AChE activity is affected by repeated low-level malathion administration, female Wistar rats were exposed to malathion (50 and 100 mg/kg, intraperitoneally) for 3 consecutive days. Exposure to malathion 50 mg/kg did not affect AChE activity, as previously observed. Contrary to expectation, 100 mg/kg malathion produced a significant increase in AChE activity in both cerebral cortex and hippocampus. Besides AChE inhibition, malathion may act as a pro-oxidative agent by interfering with antioxidant defences, as shown by a decrease of glutathione peroxidase and glutathione reductase activity in the cerebral cortex (100 mg/kg malathion). These effects are in contrast to response in the hippocampus where the increase in AChE activity correlates positively with the antioxidant defences, while the opposite was found in the cerebral cortex. These data indicate that, with low doses, and after a short period of exposure, malathion induces an up-regulation of AChE activity, a pattern similar to that found in the hippocampus for the antioxidant defences studied. The cerebral cortex was more vulnerable to malathion, as reflected in a decrease of two antioxidant enzymes. This study indicates that (i) alternatively to AChE inhibition, interference with the antioxidant defence system may be another important target for malathion toxicity; (ii) hippocampal and cortical AChE activity in rats can be increased after repeated low-dose malathion exposure. This response suggests the occurrence of a pathophysiological response in order to maintain the homeostasis of the cholinergic system in these cerebral structures.

Folic acid administration prevents ouabain-induced hyperlocomotion and alterations in oxidative stress markers in the rat brain.

OBJECTIVE Bipolar disorder (BD) is a chronic, prevalent, and highly debilitating psychiatric illness. Folic acid has been shown to have antidepressant-like effects in preclinical and clinical studies and has also been suggested to play a role in BD. The present work investigates the therapeutic value of folic acid supplementation in a preclinical animal model of mania induced by ouabain. METHODS Male Wistar rats were treated twice daily for seven days with folic acid (10, 50, and 100 mg/kg, p.o.) or the mood stabilizer lithium chloride (LiCl) (45 mg/kg, p.o.). One day after the last dose was given, the animals received an i.c.v. injection of ouabain (10 microM), a Na(+),K(+)-ATPase-inhibiting compound. Locomotor activity was assessed in the open-field test. Thiobarbituric acid-reactive substance (TBARS) levels, glutathione peroxidase (GPx), and glutathione reductase (GR) activities were measured in the cerebral cortex and hippocampus. RESULTS Ouabain (10 microM, i.c.v.) significantly increased motor activity in the open-field test, and seven days of pretreatment with folic acid (50 mg/kg, p.o.) or LiCl (45 mg/kg, p.o.) completely prevented this effect. Ouabain treatment elicited lipid peroxidation (increased TBARS levels) and reduced GPx activity in the hippocampus. GR activity was decreased in the cerebral cortex and hippocampus. These effects were prevented by pretreatment with folic acid and LiCl. CONCLUSIONS Our results show that folic acid, similarly to LiCl, produces a clear antimanic action and prevents the neurochemical alterations indicative of oxidative stress in an animal model of mania.

Complex methylmercury-cysteine alters mercury accumulation in different tissues of mice

Methylmercury (MeHg) can cause deleterious effects in vertebrate tissues, particularly in the central nervous system. MeHg interacts with sulfhydryl groups from low and high molecular weight thiols in the blood, which can facilitate MeHg uptake into different tissues. The purpose of this study was to examine the effect of MeHg-Cysteine (MeHg-Cys) complex administration on Hg-uptake in cerebral areas (cortex and cerebellum), liver and kidney of adult mice. Animals were divided into four groups: control (1 mL/kg distilled water), MeHg (2 mg/kg), Cys (2 mg/kg) and MeHg-Cys complex (0.8 molar ratio). Mice received one intraperitoneal injection per day for 60 consecutive days. Treatment with MeHg significantly increased mercury concentrations in all tissues analysed when compared with the control group. The accumulation of mercury in brain and in liver was further increased in animals that received MeHg-Cys complex when compared with the MeHg alone group. However, renal Hg decreased in MeHg-Cys treated mice, when compared with the group treated only with MeHg. In summary, the transport of MeHg-Cys complex was tissue-specific, and we observed an increase in its uptake by liver and brain as well as a decrease in kidney.

Effects of Hg(II) Exposure on MAPK Phosphorylation and Antioxidant System in D. melanogaster

The heavy metal mercury is a known toxin, but while the mechanisms involved in mercury toxicity have been well demonstrated in vertebrates, little is known about toxicological effects of this metal in invertebrates. Here, we present the results of our study investigating the effects associated with exposure of fruit fly Drosophila melanogaster to inorganic mercury (HgCl2). We quantify survival and locomotor performance as well as a variety of biochemical parameters including antioxidant status, MAPK phosphorylation and gene expression following mercury treatment. Our results demonstrate that exposure to Hg(II) through diet induced mortality and affected locomotor performance as evaluated by negative geotaxis, in D. melanogaster. We also saw a significant impact on the antioxidant system including an inhibition of acetylcholinesterase (Ache), glutathione S‐transferase (GST) and superoxide dismutase (SOD) activities. We found no significant alteration in the levels of mRNA of antioxidant enzymes or NRF‐2 transcriptional factor, but did detect a significant up regulation of the HSP83 gene. Mercury exposure also induced the phosphorylation of JNK and ERK, without altering p38MAPK and the concentration of these kinases. In parallel, Hg(II) induced PARP cleavage in a 89 kDa fragment, suggesting the triggering of apoptotic cell death in response to the treatment. Taken together, this data clarifies and extends our understanding of the molecular mechanisms mediating Hg(II) toxicity in an invertebrate model.